Diagnostic imaging methods using rhenium and technetium complexes containing a hypoxia-localizing moiety

ABSTRACT

Novel methods, processes and metal complexes attached to a hypoxia-localizing moiety comprising a metal, preferably radionuclide of rhenium or technetium; a hypoxia-localizing moiety; and, a complexing ligand, wherein said ligand and said radionuclide combined have cell membrane permeabilities greater than that of sucrose, are disclosed.

This is a divisional of application Ser. No. 08/415,743, filed Apr. 3,1995, which is a continuation of 08/054,120, filed Apr. 27, 1993, nowabandoned which is a continuation-in-part of 07/976,079, filed on Nov.13, 1992, now abandoned, which is a continuation-in-part of 07/784,486,filed on Oct. 29, 1991, now abandoned.

BACKGROUND OF THE INVENTION

Many of the procedures presently conducted in the field of nuclearmedicine involve radiopharmaceuticals which provide diagnostic images ofblood flow (perfusion) in the major organs and in tumors. The regionaluptake of these radiopharmaceuticals within the organ of interest isproportional to flow; high flow regions will display the highestconcentration of radiopharmaceutical, while regions of little or no flowhave relatively low concentrations. Diagnostic images showing theseregional differences are useful in identifying areas of poor perfusion,but do not provide metabolic information of the state of the tissuewithin the region of apparently low perfusion.

There is a need for new radiopharmaceuticals which specifically localizein hypoxic tissue, i.e., tissue which is deficient in oxygen, but stillviable. These compounds should be retained in regions which are hypoxic,but should not be retained in regions which are normoxic. Aradiopharmaceutical with these properties will display relatively highconcentrations in such hypoxic regions, with low concentrations innormoxic and infarcted regions.

Diagnostic images with this radiopharmaceutical should readily allow theidentification of tissue which is at risk of progressing to infarction,but still salvagable in, for example, the heart and brain.

It is well known that tumors often have regions within their mass whichare hypoxic. These result when the rapid growth of the tumor is notmatched by the extension of tumor vasculature. A radiopharmaceuticalwhich localizes preferentially within regions of hypoxia could also beused to provide images which are useful in the diagnosis and managementof therapy of tumors as suggested by Chapman, "Measurement of TumorHypoxia by Invasive and Non-Invasive Procedures--A Review of RecentClinical Studies", Radiother. Oncol., 20(S1), 13-19 (1991).Additionally, a compound which localizes within the hypoxic region oftumors, but is labeled with a radionuclide with suitable α- orβ-emissions could be used for the internal radiotherapy of tumors.

As reported by Martin et al. ("Enhanced Binding of the Hypoxic CellMarker ³ H! Fluoromisonidazole", J. Nucl. Med., Vol. 30, No. 2, 194-201(1989)) and Hoffman et al. ("Binding of the Hypoxic Tracer H-3!Misonidazole in Cerebral Ischemia", Stroke, Vol. 18, 168 (1987)),hypoxia-localizing moieties, for example, hypoxia-mediatednitroheterocyclic compounds (e.g., nitroimidazoles and derivativesthereof) are known to be retained in hypoxic tissue. In the brain orheart, hypoxia typically follows ischemic episodes produced by, forexample, arterial occlusions or by a combination of increased demand andinsufficient flow. Additionally, Koh et al., ("Hypoxia Imaging of TumorsUsing F-18!Fluoronitroimidazole", J. Nucl. Med., Vol. 30, 789 (1989))have attempted diagnostic imaging of tumors using a nitroimidazoleradiolabeled with ¹⁸ F. A nitroimidazole labeled with ¹²³ I has beenproposed by Biskupiak et al. ("Synthesis of an (iodovinyl)misonidazolederivative for hypoxia imaging", J. Med. Chem., Vol. 34, No. 7,2165-2168 (1991)) as a radiopharmaceutical suitable for use withsingle-photon imaging equipment.

While the precise mechanism for retention of hypoxia-localizingcompounds is not known, it is believed that nitroheteroaromaticcompounds, such as misonidazole, undergo intracellular enzymaticreduction (for example, J. D. Chapman, "The Detection and Measurement ofHypoxic Cells in Tumors", Cancer, Vol. 54, 2441-2449 (1984)). Thisprocess is believed to be reversible in cells with a normal oxygenpartial pressure, but in cells which are deficient in oxygen, furtherreduction can take place. This leads to the formation of reactivespecies which bind to or are trapped as intracellular components,providing for preferential entrapment in hypoxic cells. It is necessary,therefore, for hypoxia imaging compounds to possess certain specificproperties; they must be able to traverse cell membranes, and they mustbe capable of being reduced, for example, by reductases such as xanthineoxidase.

The hypoxia imaging agents mentioned above are less than ideal forroutine clinical use. For example, the positron-emitting isotopes (suchas ¹⁸ F) are cyclotron-produced and short-lived, thus requiring thatisotope production, radiochemical synthesis, and diagnostic imaging beperformed at a single site or region. The costs of procedures based onpositron-emitting isotopes are very high, and there are very few ofthese centers worldwide. While ¹²³ I-radiopharmaceuticals may be usedwith widely-available gamma camera imaging systems, ¹²³ I has a 13 hourhalf-life (which restricts the distribution of radiopharmaceuticalsbased on this isotope) and is expensive to produce. Nitroimidazoleslabeled with ³ H are not suitable for in vivo clinical imaging and canbe used for basic research studies only.

The preferred radioisotope for medical imaging is ^(99m) Tc. Its 140 keyγ-photon is ideal for use with widely-available gamma cameras. It has ashort (6 hour) half life, which is desirable when considering patientdosimetry. ^(99m) Tc is readily available at relatively low cost throughcommercially-produced ⁹⁹ Mo/^(99m) Tc generator systems. As a result,over 80% of all radionuclide imaging studies conducted worldwide utilizethis radioisotope. To permit widespread use of a radiopharmaceutical forhypoxia imaging, it is necessary that the compound be labeled with^(99m) Tc. For radiotherapy, the rhenium radioisotopes, particularly ¹⁸⁶Re and ¹⁸⁶ Re, have demonstrated utility.

EP 411,491 discloses boronic acid adducts of rhenium dioxime andtechnetium-99m dioxime complexes linked to various nitroimidazoles.Although these complexes are believed to be useful for diagnostic andtherapeutic purposes, it would be desirable obtain higher levels of therhenium or technetium radionuclide in the targeted area, than areachieved with this class of capped-dioxime nitroimidazole complexes. Itwas demonstrated that the compounds disclosed in EP 411,491 possessreduction potentials similar to 2-nitroimidazole derivatives known tolocalize in hypoxic regions. In addition, the reduction of thesecompounds is catalyzed by xanthine oxidase. However, these compoundshave poor membrane permeability. Thus, while these compounds might beretained by hypoxic cells, delivery of these compounds to theintracellular domain of these cells may be less than ideal. In addition,the complexes described in EP 411,491 require a heating step to form thehypoxia-localizing radiolabeled compounds. It would be more convenientfor the routine use of such hypoxia-localizing radiolabeled compounds tobe able to prepare such complexes at ambient temperatures.

Radiolabeled complexes of hypoxia-localizing moieties which retain thebiochemical behavior and affinity of such moieties, which are labeled atroom temperature with a suitable, easy-to-use radionuclide, and whichare capable of providing increased amounts of the desired radionuclideto the targeted area, would be a useful addition to the art.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel ligands, metal complexesof such ligands, processes for their preparation, and diagnostic andtherapeutic methods for their use, are disclosed. In particular, metalcomplexes, e.g., technetium and rhenium complexes, which are linked to ahypoxia localizing moiety, and wherein the complex has a permeabilitythrough cell membranes greater than that of ¹⁴ C-sucrose, are disclosed.Exemplary complexes are useful as diagnostic imaging agents in the caseof technetium radionuclides and improved agents for radiotherapy in thecase of rhenium radionuclides. Suitable novel ligands to form thesecomplexes may include, but are not limited to, di-, tri- or tetradentateligands forming neutral complexes or technetium or rhenium with themetal preferably in the +5 oxidation state. Examples of such ligands arerepresented by the formulae ##STR1## where at least one R is --(A)_(p)--R₂ where (A)_(p) is a linking group and R₂ is a hypoxia localizingmoiety; and wherein the other R groups are the same, or different andare independently selected from hydrogen, halogen, hydroxy, alkyl,alkenyl, alkynyl, alkoxy, aryl, --COOR₃, ##STR2## --NH₂, hydroxyalkyl,alkoxyalkyl, hydroxyaryl, haloalkyl, arylalkyl, -alkyl-COOR₃, -alkyl-CON(R₃)₂, -alkyl-N(R₃)₂, -aryl-COOR₃, -aryl-CON(R₃)₂, -aryl-N(R₃)₂, 5- or6-membered nitrogen- or oxygen-containing heterocycle; or two R groupstaken together with the one or more atoms to which they are attachedform a carbocyclic or heterocyclic, saturated or unsaturated spiro orfused ring which may be substituted with R groups;

R₁ is hydrogen, a thiol protecting group or --(A)_(p) --R₂ ;

R3 is hydrogen, alkyl or aryl;

m=2 to 5; and,

p=0 to 20.

It should be apparent that the disulfide of Ic can be reduced to thecorresponding dithiol of Ib by known methodology prior to complexingwith a metal.

The linking group (A)_(p) can be any chemical moiety which can serve tophysically distance, or otherwise isolate, the hypoxia localizing moietyfrom the rest of the complex of formula I. This might be important ifthe hypoxia localizing moiety is likely to be inhibited in its action bythe rest of the complex. For example, in the linking group, wherein p isone, A, or the various A units in forming a straight or branched chainif p>1, are independently selected from --CH₂ --, --CHR₄ --, --CR₄ R₅--, --CH═CH--, --CH═CR₄ --, --CR₄ ═CR₅ --, --C.tbd.C--, cycloalkyl,cycloalkenyl, aryl, heterocyclo, oxygen, sulfur, ##STR3## --NH--,--HC═N--, --CR₄ ═N--, --NR₄ --, --CS--; wherein R₄ and R₅ areindependently selected from alkyl, alkenyl, alkoxy, aryl, 5- or6-membered nitrogen or oxygen containing heterocycle, halogen, hydroxyor hydroxyalkyl.

In considering the various linking groups known in the art, it isunderstood that p could be any convenient value depending upon thedesign choices for the desired complex. Preferably, p is ≦20 and mostpreferably p≦10.

Listed below are definitions of the terms used to describe the complexesof this invention. These definitions apply to the terms as they are usedthroughout the specification (unless they are otherwise limited inspecific instances) either individually or as part of a larger group.

The terms "alkyl", "alkenyl" and "alkoxy" refer to both straight andbranched chain groups. Those groups having 1 to 10 carbon atoms arepreferred.

The term "aryl" refers to phenyl and substituted phenyl. Preferred arephenyl and phenyl substituted with 1, 2 or 3 alkyl, haloalkyl,aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxy, alkoxyalkyl,halogen, amino, hydroxy, or formyl groups.

The terms "halide", "halo" and "halogen" refer to fluorine, chlorine,bromine and iodine.

The expression "5- or 6-membered nitrogen containing heterocycle" refersto all 5- and 6-membered rings containing at least one nitrogen atom.Exemplary aliphatic nitrogen heterocyclic derivatives have the formula##STR4## wherein r is 0 or 1 and A is --O--, --N--R₆, --S-- or --CH--R₆wherein R₆ is hydrogen, alkyl, aryl or arylalkyl. Such groups includepyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, 4-alkylpiperazinyl,4-alkylpiperidinyl, and 3-alkylpyrrolidinyl groups. Also included withinthe expression "5- or 6-membered nitrogen containing heterocycle" arearomatic groups. Exemplary aromatic groups are pyrrolyl, imidazolyl,oxazolyl, pyrazolyl, pyridinyl, thiophenyl, pyridazinyl, thiazolyl,triazolyl and pyrimidinyl groups. The above groups can be linked via ahereto atom or a carbon atom.

The expression "5- or 6-membered nitrogen or oxygen containingheterocycle" refers to all 5- and 6-membered rings containing at leastone nitrogen or oxygen atom. Exemplary groups are those described aboveunder the definition of the expression "5- or 6-membered nitrogencontaining heterocycle". Additional exemplary groups are 1,4-dioxanyland furanyl.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that metal complexes having a permeability throughcell membranes greater than that of ¹⁴ C-sucrose provide enhancedproducts when linked to a hypoxia localizing moiety. Depending upon themetal used, complexes employing such hypoxia-localizingmoiety-containing ligands are useful as imaging agents, therapeuticagents, radiosensitizers and hypoxic tissue cytotoxins.

Cell permeability is a property of a cell membrane which describes themobility of extraneous molecules (permeants) within the internalstructure of the membrane (W. D. Stein, "Transport and Diffusion AcrossCell Membrane", New York Academic Press Inc. (1986); A. Kotyk, K.Janacek, J. Koryta, Biophysical Chemistry of Membrane Functions,Chichester, UK: John Wiley & Sons, (1988)). Molecules to which themembrane is permeable are able to penetrate through the membrane toreach the environment on the opposite side.

The examples which follow utilize a model of cell permeability based onthe studies of Audus and Borchardt ("Bovine Brain MicrovesselEndothelial Cell Monolayers as a Model System for the Blood-BrainBarrier", Ann. New York Accad. Sci., 1988; 9-18). The model consists ofa cultured monolayer of bovine brain endothelial cells, which form tightintercellular junctions. Transit of compounds across the monolayerreflects the ability of such compounds to cross the intact cell membersby passive, active and/or facilitated diffusion mechanisms. The rate oftransit is compared with ³ H₂ O (a highly permeable tracer) and ¹⁴C-sucrose (a non-permeable tracer). As discussed above, in accordancewith the present invention, it has been found that complexes containinga hypoxia localizing moiety and having cell permeability greater thanthat of sucrose provide benefits to diagnostic and/or therapeuticprocedures employing such complexes.

The present complexes, when used with a radioactive metal, providelevels of radionuclide within hypoxic tissue sufficient to enhancediagnostic and therapeutic methods employing such complexes.

Exemplary complexes of the present invention can be shown as ##STR5##where the R groups are as defined above, where M can be a radioactive ornon-radioactive metal which may have other ligand(s) X and/or Y in theunfilled coordination sites. For example, in the cases where M=rheniumor technetium, the ##STR6## portion can be shown as ##STR7## Anyradioactive metal can be employed in the present complexes, for example,technetium or rhenium for the complexes of Ib', and technetium for thecomplexes of Ia' Rhenium includes Re-186 and Re-188 radionuclides andmixtures thereof, and may also include Re-185 and Re-187. Technetiumincludes Tc-99m, Tc-94m and Tc-96.

Complexes of the present invention have not been heretofore disclosedand are useful in that they utilize the properties of the hypoxialocalizing group to provide imaging or treatment of hypoxic tissue at aparticular site. The complexes of the present invention wherein M istechnetium provide highly effective, relatively easy to use diagnosticimaging products which are characterized by a covalent bond between theradionuclide complex and the hypoxia localizing group whilesubstantially retaining the retention properties of the free hypoxialocalizing group. It can be appreciated that typical examples ofdiagnostic uses for the complexes of the present invention when M istechnetium include, but are not limited to, imaging of hypoxic tissue,present under pathological conditions in e.g., the heart, brain, lungs,liver, kidneys or in tumors.

In addition to being useful in imaging hypoxic tissue, the presentcomplexes can also be used as blood flow markers, i.e., for perfusionimaging. The initial distribution of the novel complexes is proportionalto blood flow and therefore imaging carried out soon afteradministration is an indicator of perfusion. A short time later, as thepresent complexes wash out of the normoxic tissue but are retained inthe hypoxic tissue, imaging of the hypoxic tissue is realized.

Additionally, the present invention provides stably bound complexes whenM is Re for radiotherapeutic indications. To the extent that hypoxictissue is known to be present in tumors, Re complexes of the presentinvention are suitable for radiotherapy. The compounds of this inventionwhen M is Re for use in radiotherapy can be injected into humans andconcentrate in hypoxic tissue. This allows for the targeting ofradionuclides to the desired sites with great specificity. It isunderstood, however, that radiotherapy will only be possible in thoseareas where a sufficient quantity of hypoxic tissue is present so as toprovide therapeutic levels of rhenium to the area needing

Examples of hypoxia localizing groups are hypoxia-mediatednitro-heterocyclic groups, (i.e., nitro-heterocyclic groups that can betrapped by hypoxia-mediated reduction of the nitro moiety). In additionto those described in the Koh et al. and Hoffman et al. referencesabove, hypoxia-localizing moieties may include those described in "TheMetabolic Activation of Nitro-Heterocyclic Therapeutic Agents", G. L.Kedderis et al., Drug Metabolism Reviews, 19(1), p. 33-62 (1988),"Hypoxia Medicated Nitro-Heterocyclic Drugs in the Radio- andChemotherapy of Cancer", G. E. Adams, et al., Biochem. Pharmacology,Vol. 35, No. 1, pages 71-76 (1986); "Structure-Activity Relationships of1-Substituted 2-Nitroimidazoles: Effect of Partition Coefficient andSidechain Hydroxyl Groups on Radiosensitization In vitro", D. M. Brownet al., Rad. Research, 90, 98-108 (1982); "Structure-ActivityRelationships in the Development of Hypoxic Cell Radiosensitizers", G.E. Adams et al., Int. J. Radiat. Biol., Vol. 35, NO. 2, 133-150 (1979);and "Structure-Activity Relationships in the Development of Hypoxic CellRadiosensitizers", G. E. Adams et al., Int. J. Radiat. Biol., Vol. 38,No. 6, 613-626 (1980). These all disclose various nitroheterocyclicmoieties suitable for incorporation into the complexes of the presentinvention and are incorporated herein by reference. These compoundscomprise a nitro-heterocyclic group which may include a sidechain,(A)_(p), which can serve as the linking group connecting thenitro-heterocyclic portion to the rest of the complex of formula I ofthis invention.

When the hypoxia localizing group is a hypoxia-mediatednitro-heterocyclic group, the linker/localizing group portion of thecomplex can be represented by ##STR8## the ring portion being a 5- or6-membered cyclic or aromatic ring, wherein

n is the total number of substitution positions available on the 5- or6-membered ring;

the one or more R₇ substituents are independently selected fromhydrogen, halogen, hydroxy, alkyl, aryl, alkoxy, hydroxy-alkyl,hydroxyalkoxy, alkenyl, arylalkyl, arylalkylamide, alkylamide,alkylamine and (alkylamine)alkyl;

X₁ can be nitrogen, oxygen, sulfur, --CR₄, --CR₇ ═, CR₇ R₇ or --CRR--;and

when (A)_(p) is absent (i.e., p=0) the nitroheterocyclic hypoxialocalizing moiety is linked to the rest of the complex of this inventionvia a nitrogen or carbon atom of the cyclic ring.

The references, above, regarding hypoxia localizing moieties serve toillustrate that the present thinking in the art is that the reductionpotential of the nitro-heterocyclic group directly affects its retentionin hypoxic tissue. The linking group, (A)_(p), may therefore be selectednot only according to its capacity to distance the hypoxia localizingmoiety from the rest of the complex, but also in accordance with itseffect on the reduction potential of the hypoxia-mediatednitro-heterocyclic group.

Preferred hypoxia a localizing moieties (shown with the linking groups)are 2-, 4- and 5-nitroimidazoles which can be represented by ##STR9##and nitrofuran and nitrothiazole derivatives, such ##STR10## Exemplarygroups (including (A)_(p) linking groups) include, but are not limitedto, ##STR11## where q=0 to 5. Most preferred are nitroimidazoles andderivatives thereof.

The ligands of formula Ia can be prepared by known methods such as thosedescribed in U.S. Pat. No. 4,615,876. For example an alkylene diamine ofthe formula ##STR12## is reacted with one equivalent of the chloro oxime##STR13## to provide the diamine monooxime ##STR14##

When the compound of formula Ia includes a hypoxia-localizing moiety(and optional linking group) on one but not both of the oxime portions,compound IV prepared as above, is reacted with ##STR15## to provide##STR16##

Alternatively, to prepare a compound of the formula Ia", a compound ofthe formula III' may be reacted with a compound of the formula II, andthe diamine monoxime formed having the structure: ##STR17## reacted witha compound of the formula III.

Compounds of formula Ia having the hypoxia localizing moiety, R₂, (andoptional linking group) on the alkylene portion ##STR18## where s=0 to 4and t=0 to 4 with the proviso that s+t is not greater than 4, can beprepared by reacting a compound of the formula ##STR19## with twoequivalents of a compound of formula III when the oxime portions are tobe identically substituted. Similarly, when the oxime portions are toinclude different substituents, a compound of formula V can be reactedwith one equivalent of a first compound of formula Ill and the so-formedintermediate can thereafter be reacted with one equivalent of a secondcompound of formula III'.

Exemplary compounds of the formula Ia also include the disubstitutedcompounds: ##STR20## which may be prepared by reacting two equivalentsof a compound of the formula III' with one equivalent of a compound ofthe formula II; and the trisubstituted compounds ##STR21## which may beprepared by reacting two equivalents of a compound of the formula III'with one equivalent of a compound of the formula V.

A novel and preferred process for preparing the compounds of formula Iais outlined below. This novel process is also useful for preparing anyalkylene diaminedioxime.

The novel process for the preparation of PnAO derivatives could easilybe adapted prepare compounds outside of the scope of disclosure by thoseskilled in the art.

The novel process involves the use of a haloketone instead of the chlorooxime of compounds III and III' shown above. Thus, in its broad aspects,the novel process involves the preparation of alkylene diaminedioximesby first reacting an alkylene diamine with two equivalents of ahaloketone and converting the so-formed diketone to the correspondingalkylene diaminedioxime. Similarly, where different oxime portions aredesired the alkylene diamine can be reacted with one equivalent of afirst haloketone and then with one equivalent of a second haloketone.The so-formed unsymmetrical diketone is converted to the correspondingdioxime by known methodology as discussed above.

For example, the diamine II of the formula ##STR22## can be reacted withthe haloketone ##STR23## where halogen can be Br, Cl, I, F, preferablyBr, to provide the diketone ##STR24## Diketone VII can be converted tothe corresponding dioxime product by known methods, e.g., treatment withO-trimethylsilyl hydroxylamine.

When each of the oxime portions of the final product are intended to bedifferent, the novel method herein involves reacting a compound offormula II with a chloro oxime of formula Ill to provide the diaminemonooxime of formula IV. The monooxime IV can thereafter be reacted withthe differently substituted haloketone VI to provide the monoketone##STR25##

Monoketone VIII can be converted to the corresponding dioxime product byknown methods as described above.

Alternatively, to provide unsymmetrical oximes the diamine of II can bereacted with one equivalent of a first haloketone of VI and theso-formed intermediate can thereafter be reacted with an equivalent of asecond haloketone of VI.

Specifically regarding the novel process to prepare products of formulaIa, a diamine of formula V can be reacted with two equivalents of thehaloketone VI to provide the diketone intermediates of the formula##STR26##

Diketone VII' can be converted to the corresponding dioxime by knownmethods as described above, to provide the corresponding products offormula la where the --(A)_(p) --R₂ group is on the alkylene portion ofthe ligand.

Unsymmetrical compounds of formula Ia can be prepared using suchstarting materials in the methodology described above, i.e., thesequential coupling of two dissimilar haloketones of VI to an alkylenediamine of II or V.

Similarly, a compound of formula IV can be reacted with a compound ofthe formula ##STR27## where R'=R with the proviso that one of the R'groups must be --(A)_(p) --R₂, e.g., ##STR28## to provide, in the caseusing VI'a, the corresponding ketone-oxime ##STR29## (where one of theR' groups must be --(A)_(p) --R₂)

Ketone-oxime IX can be converted to the dioxime of Ia" using knownmethodology as shown above.

To prepare the compounds of the formula ##STR30## a compound of theformula ##STR31## (prepared as described in WO 89 10759 to Mallinckrodt)can be coupled with a compound of the formula

    XI L--(A).sub.p --R.sub.2

where L is a leaving group, e.g., halogen, to provide ##STR32##

The tertiary amine disulfide of formula XII can thereafter be reduced tothe desired dithiol product of formula Ib' (where R₁ =H) using knowndisulfide reducing agents, e.g., tris(2-carboxyethyl)phosphine,dithiothreitol, and the like, as disclosed for example in theaforementioned WO 89 10759. Alternatively, the disulfide X can bereduced to the dithiol form prior to coupling with compound XI. In thiscase, standard sulfide protection should be employed prior to couplingwith XI.

To prepare the compounds of the formula ##STR33## a compound of formulaV can be reacted with a compound of the formula ##STR34## (prepared asdescribed in Kung et al., "Synthesis and Biodistribution of NeutralLipid-soluble Tc-99m Complexes that Cross the Blood-Brain-Barrier", J.Nucl. Med., 25, 326-332 (1984)) to provide compounds of the formula##STR35##

Treatment of compound XIV with a reducing agent, e.g., sodiumborohydride, provides intermediates of the formula ##STR36## which canbe reduced to the corresponding disulfide products of Ib" using knownsulfide reducing agents as discussed above.

Compounds of the formula ##STR37## where Z and/or W are --(A)_(p) --R₂and the other of Z and W can be R, can be prepared using known peptidecoupling methodology. For example, a compound of the formula ##STR38##can be coupled with a compound of the formula ##STR39## to provideintermediates of the formula ##STR40##

Intermediate XVIII can thereafter be coupled with a compound of theformula ##STR41## wherein Z and W are as defined above in formula Ib"',to provide ##STR42##

Reduction of compound XX, e.g., by treatment with borane, providescompounds of Ib"' having the following structure ##STR43##

In all of the above reactions described for preparing compounds of thisinvention, it should be readily apparent to those skilled in the artthat sulfur groups, amine groups and ketone groups may need to beprotected during the various reactions and that the so-protectedresulting products can thereafter be deprotected by known techniques.

All of the examples and the process description below where M is rheniuminvolve the use of "carrier rhenium"except as otherwise noted. Thephrase "carrier rhenium"means that the rhenium compounds used containnon-radioactive rhenium at concentrations of >10⁻⁷ M.

Preparation of the complexes of this invention wherein M is rhenium canbe accomplished using rhenium in the +5 or +7 oxidation state. Examplesof compounds in which rhenium is in the Re(VII) state are NH₄ ReO₄ orKReO₄. Re(V) is available as, for example, ReOCl₄ !(NBu₄), ReOCl₄!(AsPh₄), ReOCl₃ (PPh₃)₂ and as ReO₂ (pyridine)₄.sup.⊕. Other rheniumreagents known to those skilled in the art can also be used.

Preparation of the complexes of this invention wherein M is technetiumcan best be accomplished using technetium in the form of thepertechnetate ion. For Tc-99m, the pertechnetate ion can best beobtained from commercially available technetium-99m parent-daughtergenerators; such technetium is in the +7 oxidation state. The generationof the pertechnetate ion using this type of generator is well known inthe art, and is described in more detail in U.S. Pat. Nos. 3,369,121 and3,920,995. These generators are usually eluted with saline solution andthe pertechnetate ion is obtained as the sodium salt. Pertechnetate canalso be prepared from cyclotron-produced radioactive technetium usingprocedures well known in the art.

The formation of the technetium complexes proceeds best if a mixture ofpertechnetate ion in normal saline is mixed with the appropriate ligandcontaining at least one R group of the form --(A)_(p) --R₂ where (A)_(p)is a linking group and R₂ is a hypoxia-localizing moiety. An appropriatebuffer or physiologically acceptable acid or base may be used to adjustthe pH to a value suitable for labeling the ligand. This will varydependent upon the nature of the ligand; for example, for ligands oftype Ia, a pH in the range between ˜5.5 to ˜9.5 should be used, andpreferably a pH value in the range 7.0-8.5. For ligands of the type IIb,a pH value in the range 3-8 should be used, with a pH of ˜6.0 beingpreferred. A source of reducing agent is then added to bring thepertechnetate down to the oxidation state of Tc(V) for chelation withthe ligand. Stannous ion is the preferred reducing agent, and may beintroduced in the form of a stannous salt such as stannous chloride,stannous fluoride, stannous tartrate, stannous diethylenetriaminepentaacetic acid or stannous citrate, but other suitable reducing agentsare known in the art. The reaction is preferably run in an aqueous oraqueous/alcohol mixture, at or about room temperature, using a reactiontime of about 1 minute to about 1 hour. The reducing agent should bepresent at a concentration of 5-50 μg/ml. The ligand should optimally bepresent in a concentration of 0.5-2 mg/ml.

Alternatively, the technetium complexes of this invention can beprepared by ligand exchange. A labile Tc(V) complex is prepared by thereduction of TcO₄ ⁻ in the presence of a ligand which forms a labiletechnetium complex, such as mannitol, the hydroxycarboxylate ligandsglucoheptonate, gluconate, citrate, malate or tartrate at a pH valuethat is appropriate for the exchange ligand in question (usually 5-8). Areducing agent such as the stannous salts described above is added,which causes the formation of a labile reduced complex of Tc with theexchange ligand. This reduced Tc complex is then mixed with the ligandcontaining --(A)_(p) --R₂ at an appropriate pH value (as describedabove). The labile exchange ligand is displaced from the metal by theligand containing the hypoxia-localizing moiety, thus forming thedesired technetium complexes of this invention.

It is convenient to prepare the complexes of this invention at, or near,the site where they are to be used. A single, or multi-vial kit thatcontains all of the components needed to prepare the complexes of thisinvention (other than the Rhenium or Technetium ion) is an integral partof this invention.

A single-vial kit would contain ligand, a source of stannous salt, orother pharmaceutically acceptable reducing agent, and be appropriatelybuffered with pharmaceutically acceptable acid or base to adjust the pHto a value as indicated above. It is preferred that the kit contents bein the lyophilized form. Such a single vial kit may optionally containexchange ligands such as glucoheptonate, gluconate, mannitol, malate,citric or tartaric acid and can also contain reaction modifiers, such asdiethylenetriaminepentaacetic acid or ethylenediamine tetraacetic acid.Additional additives, such as solubilizers (for example α-, β- orγ-cyclodextrin), antioxiants (for example ascorbic acid), fillers (forexample, NaCl) may be necessary to improve the radiochemical purity andstability of the final product, or to aid in the production of the kit.

A multi-vial kit could contain, in one vial, the ingredients exceptpertechnetate that are required to form a labile Tc(V) complex asdescribed above. The quantity and type of ligand, buffer pH and amountand type of reducing agent used would depend highly on the nature of theexchange complex to be formed. The proper conditions are well known tothose that are skilled in the art. Pertechnetate is added to this vial,and after waiting an appropriate period of time, the contents of thisvial are added to a second vial that contains a source of the ligandcontaining the hypoxia-localizing moiety, as well as buffers appropriateto adjust the pH to its optimal value. After a reaction time of about 1to 60 minutes, the complexes of the present invention are formed. It isadvantageous that the contents of both vials of this multi-vial kit belyophilized. As described for the single vial kit, additional additivesmay be necessary to improve the radiochemical purity and stability ofthe final product, or to aid in the production of the kit.

Alternatively, the multi-vial kit may contain a source of ligandcontaining the hypoxia localizing moiety in one vial and a source ofstannous ion in the second vial. Pertechnetate is added to the vialcontaining ligand, and then the contents of the second vial are added toinitiate labeling. As above, the quantity and type of ligand, buffer pHand reducing agent used would depend on the nature of thehypoxia-localizing ligand and reducing agent used. Again, it isadvantageous that the contents of both vials be lyophilized.

The complexes of this invention can be administered to a host by bolusor slow infusion intravenous injection. The amount injected will bedetermined by the desired uses, e.g. to produce a useful diagnosticimage or a desired radiotherapeutic effect, as is known in the art.

Preferred complexes of this invention are those wherein the hypoxialocalizing moiety is a hypoxia-mediated nitro-heterocyclic group. Mostpreferred are those wherein the hypoxia localizing moiety is2-nitroimidazole or a derivative thereof.

In the complexes of the present invention the preferred values for(A)_(p) are alkyl, oxa-alkyl, hydroxyalkyl, hydroxyalkoxy, alkenyl,arylalkyl, arylalkylamide, alkylamide, alkylamine and (alkylamine)alkyl.

The most preferred values for (A)_(p) are selected from --(CH₂)--₁₋₅,--CH₂ --CH═CH--CH₂ --, ##STR44## --(A₃ --O--A₃ ')₁₋₃ and --(A₃ --NH--A₃')₁₋₃ ; wherein A₃ and A₃ ' are the same or different alkyl.

Preferred complexes are ##STR45## where M₁ is technetium and M₂ istechnetium or rhenium and wherin at least one R group is --(A)_(p) --R₂.

The following examples are specific embodiments of this invention.

Example 1

3,3,9,9-Tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. N-(Dimethylallyl)-2-nitroimidazole

Sodium bicarbonate (0.42 g, 50 mmol) and dimethylallyl bromide (3.28 g,22 mmol) were added to a suspension of 2-nitroimidazole (2.26 g, 20mmol) in dry acetonitrile (10 mL). The mixture was stirred under refluxfor 16 hours. The solvent was removed under reduced pressure, and theresidue was dissolved in ethyl acetate. The solution was filtered, anddried with anhydrous sodium sulfate. Removal of the solvent gave an oilwhich was recrystallized from petroleum ether (35°-50° C.). Yield 1.83g, m.p. 48°-49° C. ¹ H NMR (CDCl₃) δ7.24 (s, 1H), 7.22 (s, 1H), 5.46 (m,1H), 5.1 (d, 2H), 1.93 (s, 3H) and 1.90 (s, 3H).

B. 3-Chloro-3-methyl-1-(2-nitro-1H-imidazo-1-yl)-2-nitrosobutane

Concentrated hydrochloric acid (1 mL, 10 mmol) was added slowly to astirred suspension of the title A compound (1.81 g, 10 mmol) in isoamylnitrite (1.18 g, 10 mmol) at 0° C., with vigorous stirring. The solutionwas allowed to come to room temperature, and was stirred at thistemperature for 4-6 hours. The precipitated solid was filtered andwashed thoroughly with ethanol and dried. Yield 0.31 g, m.p. 102°-108°C. (decomp). ¹ H NMR (DMSO-d₆) δ11.9 (s, 1H), 7.25 (s, 1H), 7.05 (s,1H), 5.45 (s, 2H) and 1.8 (s, 6H).

C. N-(3-Aminopropyl)-1-amino-1,1-dimethyl-2-butanoneoxime

A suspension of 3-chloro-3-methyl-2-nitroisobutane (2.72 g, 20 mmol(prepared according to E. G. Vassian et al., Inorg. Chem., 1967;6:2043-2046)) in methanol (20 mL) was added dropwise to a solution of1,3-diaminopropane (8.8 g, 120 mmol) in dry methanol (15 mL). During theaddition of diamine, the reaction mixture was stirred at 0°-5° C. Afterthe addition, the reaction mixture was allowed to come to roomtemperature and then heated under reflux for 6 hours. Methanol wasremoved by distillation and the residue was treated with water andcooled in ice. The solid was filtered and washed with ice cold water.The filtrate was adjusted to pH 11 with 10% sodium hydroxide and thenevaporated to dryness under reduced pressure. The gummy solid wasrepeatedly extracted with isopropyl ether and the combined filtrate wascooled and filtered. The filtrate was concentrated and the oily residuewas again extracted with 1:1 hot ether/hexanes. The combined extractswere cooled and filtered again. Evaporation of the solvents gave asemi-solid which was recrystallized from hexanes/ether twice to yield acolorless solid. Yield: 1.8 g, m.p. 72°-74° C. ¹ H NMR (DMSO-d₆) δ2.6(t, 2H), 2.3 (t, 2H), 1.8 (s, 3H), 1.5 (m, 2H) and 1.2 (s, 6H).

D.3,3,9,9-Tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxide

Diethylisopropylamine 2.6 g, 20 mmol) and the title B compound (2.47 g,10 mmol) were added to a solution of the title C compound (2.0 g, 12mmol) in dry dichloromethane (15 mL). The resultant mixture was reflexedunder nitrogen for 16 hours. The reaction mixture was diluted with 15 mLof anhydrous ether and the precipitated solid was filtered andthoroughly washed with hot 1:1 ether/dichloromethane several times. Thedried solid was powdered and stirred with 25 mL of water at 5° C. for 10minutes. The insoluble material was removed by filtration and washedseveral times with ice-cold water until only one peak was observed onHPLC analysis. The product was obtained as a pale yellow solid after airdrying for several hours. Yield 1.27 g, m.p. 146°-148° C. ¹ H NMR (CD₃OD) δ7.4 (s, 1H), 7.18 (s, 1H), 5.4 (s, 2H, NI--CH₂), 2.4 (q, 4H,N--CH₂), 1.9 (s, 3H, N═C--CH₃), 1.6 (m, 2H, C--CH₂ --C), 1.35 (s, 6H,gem dimethyl) and 1.3 (s, 6H, gem dimethyl). M.S. 384 (M+H) and 401(M+NH₄).

Analysis calc'd for C₁₆ H₂₉ N₇ O₄.2.5 H₂ O: C, 47.33; H, 7.20; N, 24.15.

Found: C, 47.26; H, 7.24; N, 22.58.

Example 2

3,3,9,9-Tetramethyl-1-(4-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. N-(Dimethylallyl)-4-nitroimidazole

A solution of 4(5)-nitroimidazole (5.65 g, 50 mmol) in drydimethylformamide (10 mL) was treated with anhydrous sodium bicarbonate(8.3 g, 100 mmol) and stirred for 15 minutes. 4-Bromo-2-methyl-2-butenewas added to the reaction mixture dropwise at room temperature andstirred under nitrogen at 50°-60° C. for 16 hours. Dimethylformamide wasremoved under reduced pressure and the residue was taken up in ether(100 mL). The ether layer was washed with water and dried over anhydroussodium sulfate. Evaporation of ether left behind an oil which wasrepeatedly washed with petroleum ether (5×25 mL). The resulting pale redoil was homogeneous on TLC and was taken on to the next step withoutfurther purification. Yield: 7.95 g. ¹ H NMR (CDCl₃) δ1.7 (s, 3H, Me),1.75 (s, 3H, Me), 4.6 (d, 2H, N--CH₂), 5.4 (t, 1H, olefinic H), 7.5 (s,1H, imi H) and 7.8 (s, 1H, imi H). M.S. M+H!⁺ 182.

B. 3-Chloro-3-methyl-1-(4-nitro-1H-imidazo-1-yl)-2-nitrosobutane

A solution of the title A olefin (7.9 g, 40 mmol) and isoamyl nitrite(5.3 g, 45 mmol) in dichloromethane was cooled to 0° C. and was treatedwith dropwise addition of concentrated hydrochloric acid (5 mL, 50 mmol)keeping the reaction temperature at 0°-5° C. The reaction mixture wasstirred until all the starting olefin was consumed (by TLC,approximately 2 hours). The precipitated solid was filtered off andwashed with ethanol and dried under vacuum at room temperature for 16hours. The product was used without further purification. Yield: 0.6 g,m.p. 120°-122° C. ¹ H NMR (DMSO-d₆) δ1.9 (s, 6H, gem dimethyls), 5.18(s, 2H, N--CH₂), 7.94 (s, 1H, imi H), 8.32 (s, 1H, imi H) and 12.24 (s,1H, N--OH). M.S. M+H!⁺ 247.

C.3,3,9,9-Tetramethyl-1-(4-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

To a solution of the title C compound of Example 1 (0.356 g, 2 mmol) indry dichloromethane (5 mL), diethylisopropylamine (0.36 g, 2 mmol) wasadded followed by solid title B chloro oxime (0.446 g, 1.8 mmol) and themixture was refluxed with stirring under nitrogen for 16 hours. Thecrude product was adsorbed onto flash silica gel and chromatographed.Elution with 15:85 MeOH/CH₂ Cl₂ yielded a gum which was recrystallizedfrom isopropyl ether and acetone three times to yield a colorless solid.Yield: 0.06 g, m.p. 152°-154° C. ¹ H NMR (DMSO-d₆) δ1.17 (s, 6H, 2CH₃),1.26 (s, 6H, 2CH₃), 1.41 (m, N--CH₂ --CH₂ --N, 2H), 1.76 (s, 3H,N═C--CH₃), 2.26 (m, 4H, N--CH₂), 4.98 (s, 2H, imi N--CH₂), 7.9 (s, 1H,imi H), 8.29 (s, 1H, imi H), 10.42 (s, 1H, N--OH) and 11.63 (s, 1H,N--OH).

Analysis calc'd for C₁₆ H₂₉ N₇ O₄.H₂ O: C, 48.96; H, 7.45; N, 24.98;

Found: C, 49.11; H, 7.49; N, 24.76.

Example 3

4,4,10,10-Tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-5,9-diazadodecane-3,11-dionedioxime

A. N-(4-Methylpent-3-en-1-yl)-2-nitroindazole

To a solution of 2-nitroimidazole (3.0 g, 27 mol) in drydimethylformamide (25 mL), was added anhydrous sodium bicarbonate (4.2g, 50 mmol) followed by 5-bromo-2-methyl-2-pentene (5.0 g, 30.67 mmol).The reaction mixture was heated at 60°-70° C. with stirring undernitrogen for 16 hours. Solvent dimethylformamide and the unreactedbromide were removed under reduced pressure (<1 mm) at 50°-60° C. toyield a paste which was dissolved in water (50 mL) and extracted withethyl acetate (5×50 mL). The combined organic extracts were dried andconcentrated to give a brown oil which was recrystallized from petroleumether (b.p. to yield a yellow solid. Yield: 4.8 g, m.p. 51°-52° C. ¹ HNMR (CDCl₃) δ1.55 (s, 3H, CH₃), 1.75 (s, 3H, CH₃), 2.7 (q, 2H, olefinicCH₂), 4.5 (t, 2H, N--CH₃), 5.2 (t, 1H, olefinic H), 7.1 (s, 1H,imidazole H), 7.2 (s, 1H, imidazole H), M.S. M+H!⁺ 196, M+NH₄ !⁺ 213.

B. 4-Chloro-4-methyl-1-(2-nitro-1H-imidazo-1-yl)-3-nitrosopentane

Isoamyl nitrite (1.4 g, 12 mmol) was added to an ice-cooled solution ofthe title A olefin (2.17 g, 12 mmol) in dichloromethane (5 mL), and themixture was treated with a dropwise addition of concentratedhydrochloric acid, keeping the temperature of the reaction mixture below0° C. After stirring for an additional 2 hours, the solid formed wasisolated by filtration and washed with ice cold ethanol. The pale yellowproduct was dried under vacuum and used in the next step without furtherpurification. Yield: 1.7 g, m.p. 105°-107° C. ¹ H NMR (DMSO-d₆) δ1.7 (s,6H, gem dimethyl), 2.9 (t, 2H, oxime CH₂), 4.7 (t, 2H, N--CH₂), 7.1 (s,1H, imidazole H) and 7.5 (s, 1H, imidazole H). M.S. M+H!⁺ 261, M+NH₄ !⁺278.

C.4,4,10,10-Tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-5,9-diazadodecane-3,11-dionedioxime

Sodium bicarbonate (0.42 g, 5 mmol) was added to a solution of the titleC compound of Example 1 (0.86 g, 5 mmol) in dry tetrahydrofuran (10 mL),and then the reaction mixture was treated with the title B compound (1.3g, 5 mmol). The mixture was heated with stirring under reflux for 6hours. The solution was reduced in volume to about 5 mL and the crudeproduct was treated with 5 g of flash silica gel and then dried undervacuum to a free flowing powder. This powder was loaded on to a silicagel column and chromatographed three times. The product was eluted as alow melting solid with 9:1 dichloromethane/methanol. Yield: 0.13 g, m.p.65°-67° C. ¹ H NMR (DMSO-d₆) δ1.2 (s, 12H, 4 CH₃), 1.45 (m, 2H, 5 CH₂),1.8 (s, 3H, ═N--CH₃), 2.3 (m, 4H, N--CH₂), 2.85 (t, 2H, ═N--CH₂), 4.8(t, 2H, imidazole N--CH₂), 7.2 (s, 1H, imidazole H), 7.6 (s, 1H,imidazole H), 10.4 (s, 1H, N--OH), 10.85 (s, 1H, N--OH). M.S. M+H!⁺ 398.

Analysis calc'd for C₁₇ H₃₁ N₇ O₄ : C, 51.37; H, 7.86; N, 24.67;

Found: C, 51.89; H, 7.89; N, 23.27.

Example 4

6-Hydroxy-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. N-(3-Amino-2-hydroxypropyl)-1-amino-1,1-dimethyl-2-butanoneoxime

3-Chloro-3-methyl-2-nitrosobutane (6.75 g, 0.05 mol) was addedportionwise to a cooled (0° C.) solution of 1,3-diamino-2-hydroxypropane(14 g, 0.155 mol) in methanol (75 mL). After the addition, the reactionmixture was allowed to warm to room temperature and heated under refluxfor 12 hours. Methanol was removed on a rotary evaporator. The residuewas neutralized with methanolic ammonia. Excess methanol was removed ona rotary evaporator. The residue was dissolved in dioxane-water (2:1,300 mL) and the solution was cooled to 0° C. Sodium carbonate (31.8 g,0.3 mol) was added to this mixture followed by di-t-butyl dicarbonate(65.47 g, 0.3 mol). The reaction mixture was stirred at 0° C. for 2hours and at room temperature for 6 hours.

Dioxane and water were removed on a rotary evaporator and the residuewas poured into water and extracted with ethyl acetate. The ethylacetate solution was washed with water and dried with sodium sulfate.Ethyl acetate was removed on a rotary evaporator and the residue waschromatographed over silica gel (hexane-ethyl acetate 50: 50).Di-t-Boc-1,3-diamino-2-hydroxypropane eluted in the earlier fractions.These fractions were collected and solvent was evaporated to yield athick oil. Yield 4.9 g. This was treated with methanolic hydrochloricacid (25 mL) at room temperature for 2 hours. Methanol was removed underreduced pressure and the solid obtained was neutralized with methanolicammonia to yield the product as a white solid. This was used for thenext step without further purification. Yield: 3.98 g. ¹ H NMR (D₂ O)δ1.54 (s, 6H, C(CH₃)₂), 1.80 (s, 3H, CH₃), 2.92-3.32 (m, 4H, CH₂), 4.18(m, 1H, CHOH).

B.6-Hydroxy-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime dihydrochloride

The title B compound of Example 1 (1.6 g, 0.0065 mol) was added to aslurry of the title A compound (1.4 g, 0.0075 mol) anddiisopropylethylamine (1 g, 0.0078 mol) in acetonitrile (10 mL) and themixture was stirred at room temperature for 24 hours. Acetonitrile wasremoved on a rotary evaporator and the thick yellow oil obtained waschromatographed over silica gel (CH₂ Cl₂ :CH₃ OH, (9:1) and CH₂ Cl₂ :CH₃OH, (9:2)). Fractions containing the product were combined and solventwas evaporated to yield a thick oil. ¹ H NMR of the oil indicated thepresence of the product and diisopropylethylamine. The oil was leftunder vacuum for 12 hours. The thick oil was then triturated severaltimes with methylene choride to remove the diisopropylethylamine. Theresidue was then dissolved in water and freeze dried. Yield: 0.65 g,m.p. 114°-115° C. ¹ H NMR (D₂ O) δ1.33, 1.44 and 1.88 (s, 15H, CH₂),2.42-2.92 (m, 4H, CH₂), 3.90 (m, 1H, CHOH), 5.34 (s, 2H, CH₂ N), 7.14and 7.31 (s, 2H, C═N and C═C). M.S. calc'd 400.2308; found: 400.2298.

Example 5

3,3,9,9-Tetramethyl-6-((2-nitro-1H-imidazo-1-yl)acetamido)-4,8-diazaundecane-2,10-dionedioxime

A. (N,N'-bis-t-Boc)-2-mesyloxypropane-1,3-diamine

Methanesulfonyl chloride (6.01 g, 4.1 mL, 0.0525 mol) was added to anice-cooled (0° C.) solution of 1.3-Bis-N-t-Boc-2-hydroxypropane (14.5 g,0.05 mol) and triethylamine (6.07 g, 8.5 mL) in methylene chloride overa period of 45 minutes. The reaction mixture was then stirred at 0° C.for 1 hour and at room temperature for 12 hours. Precipitatedtriethylamine hydrochloride was removed by filtration, and the filtratewas evaporated to dryness under reduced pressure. The residue was pouredinto water. The resultant solid was isolated by filtration, air driedand used without further purification. Yield 18 g, m.p. 139°-140° C. ¹ HNMR (CDCl₃) δ1.41 (s, 18H), 3.08 (s, 3H), 3.30 (m, 2H), 3.45 (m, 2H),4.65 (m, 1H), 5.15 (bs, 2H).

B. 1.3-Bis-N-t-Boc-2-azidopropane

Sodium azide (6.5 g, 0.1 mol) was added to a solution of the title Acompound (9.2 g, 0.025 mol) in dry dimethylformamide (50 mL), and themixture was stirred at 70° C. for 12 hours. The reaction mixture wascooled and poured into water. The precipitated solid was isolated byfiltration, and was washed with water and air dried. Yield 6.45 g, m.p.90°-91° C. ¹ H NMR (CDCl₃) δ1.45 (s, 18H), 3.15 (m, 2H), 3.35 (m, 2H),3.64 (m, 1H), 5.04 (bs, 2H).

C. 1,3-Bis-N-t-Boc-1,2,3-triaminopropane

10% Palladium-on-carbon (1 g) was added to a solution of the title Bcompound (6.5 g, 0.0205 mol) in methanol (25 mL) and hydrogenated at 50psi for 12 hours. The catalyst was removed by filtration and methanolwas removed on a rotary evaporator. The resultant oil solidified onstanding. Yield 4.82 g (82%), m.p. 94°-96° C. ¹ H NMR (CDCl₃) δ1.42 (s,18H, Boc), 2.89 (m, 1H, CHNH₂), 3.12 (m, 4H, CH₂), 5.20 (m, 2H, NH).

D. 1,3-Bis-N-t-Boc-2-(2-nitroimidazol-1-yl)-acetamido-1,3-diaminopropane

Carbonydiimidazole (3.08 g, 0.019 mol) was added to a solution of2-(2-nitroimidazol-1-yl) acetic acid (3.1 g, 0.018 mol (preparedaccording to P. Webb et al., J. Lab. Cmpds. Radiopharm., 1990;28:265-271)) in dimethylformamide (25 mL). The mixture was stirred atroom temperature for 45 minutes. 1,3-Bis-N-t-Boc-2-aminopropane (5.3 g,15 0.018 mol) was added and the resultant mixture was stirred at 50° C.for 12 hours. Dimethylformamide was removed under vacuum and the residuewas treated with water. The solid which formed was isolated byfiltration and air dried. Yield 6.5 g. ¹ H NMR (CDCl₃) δ1.44 (s, 18H),2.90 (m, 1H), 3.08 (m, 4H), 5.24 (bs, 2H).

E. 2-(2-Nitroimidazol-1-yl)acetamido-1,3-diaminopropane dihydrochloride

The title D compound (6.5 g) was dissolved in methanolic hydrochloricacid (20 mL), and the reaction mixture was stirred at room temperaturefor 1 hour. The diamine dihydrochloride was precipitated by the additionof dry ether (200 mL). Yield 4.25 g. ¹ H NMR (dihydrochloride in D₂ O)δ3.08-3.35 (m, 4H), 4.51 (m, 1H), 5.31 (s, 2H), 7.19 (s, 1H), 7.43 (s,1H). ¹ H NMR (free base in D₂ O) δ3.01-3.28 (m, 4H), 4.45 (m, 1H), 5.21(s, 2H), 7.15 (S, 1H), 7.39 (s, 1H).

F.3,3,9,9-Tetramethyl-6-((2-nitro-1H-imidazo-1-yl)acetamido)-4,8-diazaundecane-2,10-dione

Sodium hydrogen carbonate (5.88 g, 0.07 mol) and2-bromo-2-methylbutan-3-one (6.1 g, 0.07 mol (prepared according to W.Pfleiderer et al., Ann. Chem., 1966; 99:3008-3021)) were added to aslurry of the title E compound (4.25 g, 0.0135 mol) in drydimethylformamide (40 mL). The reaction mixture was stirred at 45° C.for 12 hours. Methylene chloride (200 mL) was added to this reactionmixture, and the insoluble material was removed by filtration. Methylenechloride was removed on a rotary evaporator and the dimethylformamidewas removed under vacuum. The residue was chromatographed on silica geland eluted with ethyl acetate-methanol (9:1). Fractions containing theproduct were collected. Evaporation of the solvent yielded the desireddiaminediketone. Yield 2.56 g. A sample of the product was crystallizedfrom hexane, to provide product with a m.p. of 96°-97° C. ¹ H NMR (D₂ O)δ1.22 (d, 12H, C(CH₃)₂), 2.12 (s, 6H, CH₃), 2.32-2.58 (m, 4H), 3.9 (m,1H, CH), 5.21 (s, 2H), 7.15 (S, 1H), 7.39 (s, 1H). M.S. (M+H)⁺ =411.

G.3,3,9,9-Tetramethyl-6-((2-nitro-1H-imidazo-1-yl)acetamido)-4,8-diazaundecane-2,10-dionedioxime

O-Trimethylsilyl hydroxylamine (1 g, 1.22 mL, 0.01 mol) was added to asolution of the title F compound (550 mg, 0.00133 mol) in methylenechloride (2 mL). The reaction mixture was allowed to stand at roomtemperature for 24 hours. Methanol (2.0 mL) was added to the reactionmixture and the solvent was removed on a rotary evaporator. Theresultant solid was crystallized from water. Yield 329 mg, m.p. 72°-73°C. ¹ H NMR (D₂ O) δ1.12 (s, 12H, C(CH₃)₂), 1.72 (s, 6H, CH₃), 2.22-2.45(m, 4H), 3.8 (m, 1H, CH), 5.1 (s, 2H), 7.15 (s, 1H), 7.39 (s, 1H). M.S.(M+H)⁺ =441.

Analysis calc'd for C₁₈ H₃₂ N₈ O₅ : C, 49.08; H, 7.32; N, 25.44;

Found: C, 49.42; H, 7.54; N, 25.65.

Example 5a

3,3,9,9-Tetramethyl-6-((2-nitro-1H-imidazo-1-yl)ethyl)-4,8-diazaundecane-2,10-dionedioxime

A. Benzyl 2-methylsulphonyloxyethyl ether

Triethylamine (18 g, 0.178 mol) was added to a solution ofbenzyloxyethanol (25 g, 0.165 mol) in methylene chloride (200 mL). Thesolution was cooled to 0° C. and methanesulfonyl chloride (19.95 g,0.174 mol) was added dropwise over a period of 0.5 hour. After theaddition was complete the reaction mixture was stirred at 0° C. for anadditional 1 hour and at room temperature for 12 hours. The precipitatedtriethylamine hydrochloride was filtered and washed with dry ether. Thecombined filtrate and the were concentrated to a thick viscous oil (37g). ¹ H NMR (CDCl₃) δ3.15 (s, 3H, CH₃), 3.82 (t, 2H), 4.52 (t, 3H), 4.67(s, 2H) and 7.42 (m, 5H, AT-H).

B. Benzyl 2-bromoethyl ether

The title A compound (37 g, 0.16 mol) was added to a solution of lithiumbromide (86.85 g, 0.8 mol) in acetone (300 mL), and the resultingsolution was heated under gentle reflux for 12 hours. The reactionmixture was cooled and the acetone was removed on a rotary evaporator.The residue was taken up in ether and washed successively with water anddried. Evaporation of ether afforded a liquid which was distilled underreduced pressure to yield the product (32 g), b.p. 95° C./1.5mm. ¹ H NMR(CDCl₃) δ3.60 (t, 2H), 3.90 (t, 3H), 4.70 (s, 2H) and 7.46 (m, 5H,Ar--H).

C. Diethyl 1-(2-Benzyloxyethyl)malonate

Diethyl malonate (8.0 g, 0.05 mol) was added to a solution of sodiumethoxide prepared from 1.2 g (0.052 g atom) of sodium in ethanol (300mL). The title B compound (10.75 g, 0.05 mole) was added dropwise tothis solution and the reaction mixture was heated under reflux for 12hours. Ethanol was evaporated on a rotary evaporator and the residue waspoured into water and extracted with ether and dried with sodiumsulfate. Evaporation of ether gave an oil. This was distilled undervacuum to yield 9.5 g of the product, b.p. 185° C./2 mm. ¹ H NMR (CDCl₃)δ1.21 (t, 6H), 2.24 (q, 2H), 3.52 (m, 3H), 4.15 (m, 4H), 4.45 (s, 2H)and 7.31 (m, 5H, Ar--H).

D. 1-(2-Benzyloxyethyl)malonamide

The title C compound (9.0 g) was treated with ethanolic aqueous ammoniaand the reaction mixture was stirred at room temperature for 12 hours.Evaporation of the solvent gave a white solid which was crystallizedfrom water to yield the product (4.5 g), m.p. 165°-70° C. ¹ H NMR(DMSO-d₆) δ1.92 (m, 2H), 3.14 (t, 1H), 3.35 (m, 2H), 4.12 (s, 2H), 7.05(s, 2H), 7.22 (s, 2H) and 7.34 (m, 5H, Ar--H).

E. 1,3-Diamino-N,N'-di-t-Boc-2-benzyloxyethylpropane

BH₃ -THF complex (1M, 750 mL) was added to a slurry of the title Dcompound (28.0 g, 0.118 mol) in dry tetrahydrofuran (500 mL) over aperiod of 1 hour and the reaction mixture was stirred at roomtemperature for 48 hours. Excess borane was decomposed by the dropwiseaddition of water. Dilute hydrochloric acid was added until the solutionbecame acidic. Tetrahydrofuran was removed on a rotary evaporator. Theresidue was suspended in dioxane-water (2:1, 500 mL). Sodium carbonate(31.8 g, 0.3 mol) was added and the mixture was cooled to 0° C.Di-t-butyl dicarbonate (58.9 g, 0.27 mol) was added and the mixture wasstirred at 0° C. for 2 hours and at room temperature for 12 hours.Dioxane-water was removed on a rotary evaporator and the residue wastreated with water. The crude product was extracted with ethyl acetate,and the extract was dried over sodium sulfate. Ethyl acetate was removedon a rotary evaporator and the thick oil obtained was chromatographedover silica gel (hexane:ethyl acetate, 7:3) to yield 27 g of the title Ecompound. ¹ H NMR (CDCl₃) δ1.41 (s, 18H, tBoc), 1.52 (m, 2H, CHCH), 1.72(m, 1, CH), 2.9-3.2 (m, 4H, CH(CH₂ -NHtBoc)₂), 3.6 (m, 1H, OCH₂), 4.5(s, 2H, PhH₂), 5.2 (m, 2H, NH), 7.3 (m, 5H, ArH).

F. 1,3-Diamino-N,N'-di-t-Boc-2-hydroxyethylpropane

Palladium on carbon (10%, 1 g) was added to a solution of the title Ecompound (7.5 g) in methanol (50 mL) and hydrogenated at 50 psi for 24hours. Methanol was removed on a rotary evaporator,1,3-bis-N-t-butyloxycarbonyl-2 (2-hydroxyethyl)propane was obtained as awhite solid (5 g), m.p. 101°-02° C. ¹ H NMR (CDCl₃) δ1.45 (s, 18H,tBoc), 1.65 (m, 1H, CH), 2.9-3.2 (m, 6H, CH(CH₂ NHtBoc)₂ and CH₂ CH),3.78 (m, 2H, OCH₂), 5.2 (m, 2H, NH).

G. 1,3-Diamino-N,N'-di-t-Boc-2-mesyloxyethylpropane

Triethylamine (1.36 g, 1.89 mL, 0.0134 mol) was added to a solution ofhydroxyethyl derivative (3.5 g, 0.0112 mol) in methylene chloride wasadded and the mixture was cooled to 0° C. Methanesulfonyl chloride (1.43g, 0.0125 mol) was added slowly over a period of 0.5 hour and thereaction mixture was stirred at 0° C. for 1 hour and at room temperaturefor 12 hours. Methylene chloride was removed and the solid obtained wascrystallized from hexane to yield 3.9 g of the title G compound, m.p.109°-10° C. ¹ H NMR (CDCl₃) δ1.41 (s, 18H, t. Boc), 1.52 (m, 2H, CH₂CH), 1.72 (m 1H, OH), 3.0 (s, 3H, CH₃), 3.1 (m, 4H, CH(CH₂ NHtBoc)₂),4.45 (m, 2H, OCH₂), 5.15 (m, 2H, NH).

H. 2-Bromoethyl-1,3-diamino-N,N'-di -t-Boc propane

A solution of the title G compound (1.98 g, 0.5 mol) and lithium bromide(4.34 g, 0.05 mol) in acetone (50 mL) was stirred at room temperaturefor 24 hours. Acetone was removed on a rotary evaporator and the title Hcompound as obtained as an oil (1.5 g). This product was used withoutfurther purification.

I. 1,3-Diamino-N,N'-di-t-Boc-2-(2-(2-nitro-1H-imidazo-1-yl)ethylpropane

Sodium hydride (0.12 g, 0.005 moL) was added to a suspension of2-nitroimidazole (0.56 g, 0.005 mol) in dry acetonitrile (5 mL), and themixture was stirred at room temperature for 15 minutes. Acetonitrile wasremoved under vacuum and the residue was dissolved in drydimethylformamide (5.0 mL). The title H compound (1.14 g, 0.003 mol wasadded to the dimethylformamide solution and the mixture was heated in anoil bath at 110° C. for 2 hours. The mixture was cooled, anddimethylformamide was removed under vacuum. The residue was treated withwater and extracted with methylene chloride. The methylene chloridesolution was separated, dried over sodium sulfate, and solvent wasremoved on a rotary evaporator. The crude product was chromatographedover silica gel (hexane:ethyl acetate, 50:50). The fractions containingthe product were collected and evaporated to afford the product as athick yellow oil which solidified on standing. Yield 0.52 g. ¹ H NMR(CDCl₃) δ1.35 (s, 18H, Boc)0 1.6 (m, 2H, CH(CH₂ CH₂ N), 3.12 (m, 5H,CH(CH₂ NH)₂ and CH), 4.50 (t, 2H, CH(CH₂ CH₂ N), 5.12 (m, 2H, NH), 7.0and 7.25 (s, 2H, CH═CH).

J.3,3,9,9-Tetramethyl-6-((2-nitro-1H-imidazo-1-yl)ethyl)-4,8-diazaundecane-2,10-dione

The t-Boc protecting groups were removed from the title I compound bytreatment with methanolic hydrochloric acid (2 mL). Methanol was removedunder vacuum to afford the dihydrochloride. 1H NMR (D₂ O) δ2.0 (m, 2H,CH(CH₂ CH₂ N), 2.20 (m, 1H, CH), 3.12 (d, 4H, CH(CH₂ NH), 4.50 (t,CH(CH₂ CH₂ N), 7.10 and 7.42 (s, 2H, CH═CH). The dihydrochloride wasneutralized with ethanolic ammonia and the diamine free base obtainedwas used as such without further purification.

3-Bromo-3-methylbutan-2-one (0.5 g, 3.0 mmol) was added to a mixture ofthe diamine (0.2 g, 1 mmol) and sodium bicarbonate (0.25 g, 3.0 mmol) indimethylformamide (2.0 ml ) and the mixture was stirred at 50° C. for 24hours. Dimethylformamide was removed under vacuum and the crude productwas chromatographed over silica gel (CH₂ Cl₂ :CH₃ OH, 9:1, 8:2).Fractions containing the product were collected and evaporated to givethe title J compound (110 mg) as a thick oil. NMR (D=O) δ1.33 (d and m,13H, C(CH₃) and 1.80 (m, 2H, CH(CH₂ CH₂ N), 2.19 (s, 6H, CH₃), 2.65 (m,4H, CH(CH₂ NH), 4.40 (t, 2H, CH(CH₂ CH₂ N), 7.10 and 7.39 (s, 2H,CH═CH).

K.3,3,9,9-Tetramethyl-6-((2-nitro-1H-imidazo-1-yl)ethyl)-4,8-diazaundecane-2,10-dionedioxime

Diketone (65 mg) was dissolved in dry methylene chloride (0.5 ml) andtreated with trimethylsilyl hydroxylamine (0.3 mL). The reaction mixturewas heated under reflux for 24 hours. Methylene chloride was removed andthe residue was treated with methanol. Evaporation of methanol affordedthe product as a thick paste which was dissolved in water and freezedried to yield 62 mg of the title K compound, m.p. 174°-76° C. ¹ H NMR(D₂ O) δ1.2 (d and m, 13H, C(CH₃) and CH), 1.75 (s and m, 8H, CH(CH₂ CH₂N) and CH₃), 2.55 (m, 4H, CH(CH₂ NH), 4.36 (t, 2H, CH(CH₂ CH₂ N), 7.06and 7.34 (s, 2H, CH═CH), MS: (M+H)⁺ =412⁺.

Analysis calc'd for C₁₈ H₃₅ N₇ O₄.4H₂ O: C, 44.70; H, 7.30; N,20.29;

Found: C, 45.08; H, 7.14; N, 20.18.

Example 5b

5,8-Diaza-1,2-dithia-5-(2-(2-nitro-1H-imidazo-1-yl)ethyl)-3,3,10,10-tetramethylcyclodecane

A. 5,8-Diaza-1,2-dithia-3,3,10,10-tetramethylcyclodecane

Sodium borohydride (9.12 g, 0.24 mole) was added in portions at roomtemperature with stirring over a period of about 2 hours to a solutionof 5,8-diaza-1,2-dithia-5-3,3,6,6-tetramethylcyclodeca-4,8-diene (9.2 g,40 mmol, reported by H. F. Kung, M. Molnar, J. Billings, R. Wicks, M.Blau, "Synthesis and Biodistribution of Neutral Lipid-Soluble Tc-99mComplexes that Cross the Blood-Brain-Barrier", J. Nucl. Med., 1984;25:326-332) in ethanol (500 mL). The reaction mixture stirred at roomtemperature for an additional 20 hours. Ethanol was removed underreduced pressure and the crude product was chromatographed over a flashsilica gel column. Elution with 9:1 dichloromethane/methanol furnishedthe cyclized product (described by S. Z. Lever, "Correction: Design,Preparation and Biodistribution of a Technetium-99m TriaminedithiolComplex to Assess Regional Cerebral Blood Flow", J. Nucl. Ned., 1987;28:1064-1065) followed by the required diamine on continued elution with9:1:0.1 dichloromethane/methanol/ammonia. The product was recrystallizedfrom petroleum ether to yield a colorless solid. Yield: 0.66 g, m.p.58°-60° C.

B.5,8-Diaza-1,2-dithia-5-(2-(2-nitro-1H-imidazol-yl)ethyl!-3,3,10,10-tetramethylcyclodecane

Potassium fluoride on celite (0.82 g, 14.1 mmol) was added to a solutionof the title A compound (0.66 g, 2.82 mmol) in dry acetonitrile (10 mL),and the reaction mixture was stirred for 5 minutes. Bromoethylnitroimidazole (0.65 g, 2.82 mmol, described by D.C. Heimbrook, K.Shyam, A. C. Sartorelli, "Novel 1-haloalkyl-a-nitroimidazoleBioreductive Alkylating Agents", Anti-Cancer Drug Design, 1988,2:339-350) was added and stirred under nitrogen and under reflux for 16hours. Additional bromoethyl nitroimidazole (0.22 g, 1 mmol) was addedfollowed by potassium fluoride on celite (0.3 g, 5 mmol) and stirringwith reflux was continued for another 24 hours. Solvent was removedunder reduced pressure and the residue was treated with 20 mL of water.The pH of the solution was adjusted with sodium bicarbonate to ≧8. Thesolution was extracted with dichloromethane (5×20 mL). The combinedorganic layer was washed with water and dried with anhydrous sodiumsulfate. Removal of the solvent gave a semi-solid which waschromatographed over flash silica gel. Elution with 5% methanol indichloromethane furnished an oil which was homogeneous on TLC. Yield:0.065 g. ¹ H NMR (CDCl₃) δ1.1, 1.3, 1.35 and 1.45 (4s, 12H, gemdimethyls), 2.5-3.2 (m, 10H, N--CH₂), 3.9 (bs, 1H, NH), 4.5 (m, 2H, imiCH₂), 7.1 (s, 1H, imid H) and 7.4 (s, 1H, imi H). M.S. M+H!⁺ =374. TLC(9:1, dichloromethane/methanol, silica gel ): R_(f) 0.38. HPLC: Singlepeak, R_(t) =10.06 min, with UV detection (230 nm) with a Dynamax C₁₈column, 25 cm×0.46 cm, and gradient elution with acetonitrile and water(containing 0.1% trifluoroacetic acid).

Example 6

⁹⁹ Tc!Oxo3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioximato!-(3-)-N,N',N",N"'!technetium(V)

NH₄ ⁹⁹ TcO₄ (26.6 mg, 0.148 moles) was dissolved in saline (4 mL). Thetitle compound from Example 1 (86.4 mg, 0.225 mmoles) was dissolved insaline (10 mL) containing 10 drops 3M hydrochloric acid, and the pH ofthe solution adjusted to 6.3 with sodium hydroxide solution. Thesolutions of ligand and pertechnetate were combined. 0.1M Sodiumhydrogen carbonate (5 mL) was added and the pH was adjusted to pH8.5-9.0 with potassium hydroxide. Diethyl ether (60 mL) was added,followed by a dropwise addition of a suspension of stannous tartrate(83.6 mg, 0.313 mmol) in saline (5 mL). The reaction mixture was stirredfor 10 minutes. The ether layer was separated and the aqueous layerextracted with several aliquots of ether (until the yellow color ofproduct was no longer observed in the ether layer). The combined etheraliquots (110 mL) were dried over anhydrous sodium sulfate, and reducedto 2 mL by rotary evaporation. The product was purified by silica gelcolumn chromatography, using ether as eluent. Solvent was removed to avolume of ˜1 mL, and stored overnight in a -18° C. freezer. Mediumorange crystals were obtained. These were separated by filtration,washed with cold ether, and vacuum dried for four hours. Yield: 25.8 mg.¹ H NMR (CD₂ Cl₂) δ1.39-1.49 (m, 12H, C(CH₃)₂), 1.73-1.77 (m, 1H, CH),2.33 (s, 3H, CH₃), 2.35-2.41 (m, 1H, CH), 3.34-3.40 (m, 2H, CH₂),3.46-3.51 (m, 2H, CH₃), 5.63-5.73 (m, 2H, CH₂), 7.09 (s, 1H, imidazoleCH), 7.47 (s, 1H, imidazole CH). M.S.: (M+H)⁺ =496, (M--H)⁻ =494.

Analysis calc'd for C₁₆ H₂₆ N₇ O₅ Tc: C, 38.79; H, 5.29; N, 19.79;

Found: C, 39.21; H, 5.60; N, 19.47.

Example 6a

⁹⁹ Tc!Oxo3,3,9,9-tetramethyl-1-(4-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioximato !-(3-)-N,N',N", N"'!technetium (V)

To a stirring solution of N(butyl)₄ !TcOCl₄ ⁻ (45.5 mg, 0.091 mmol)(prepared by the method of F. A. Cotton. A. Davison, V. Day et al.,Inorg. Chem., 1979, 18, 3024) was added 1 mL of methanol and 120 mL ofneat ethylene glycol, followed by 1.2 mL of 0.75M sodium acetate inmethanol. Addition of the ligand of Example 2 (namely3,3,9,9-tetramethyl-1-(4-nitro-1H-imidazo-1-yl)4,8-diazeundecane-2,10-dionedioxime (53.6 mg, 0.14 mmol) caused the purple solution to turn deepyellow orange. After 3 minutes, 10 mL of methylene chloride was added,and the reaction was stripped to an orange oil by rotary evaporation.The complex was purified by passage through a silica gel column that wasconditioned and eluted with methylene chloride. The red-orange band wasevaporated to an oil, triturated to a solid with 15 mL of hexanes, andthe solid was isolated and dried in vacuo overnight to yield 30.3 mg ofthe title compound. M.S.: (M+H)⁺ =496; (M+H-4-nitroimidazole)⁺ =383;(M--H)⁻ =494. ¹ H NMR (C₆ D₆): δ1.4-1.6 (m, 12H, CH₃), 1.75 (m, 1H, CCH₂C), 2.4 (m, 1H, CCH₂ C), 2.34 (s, 3H, CH₃ C═N), 3.35 (t, 1H, NCH₂), 3.5(m 1H, NCH₂), 4.9 (d, 1H, imidazole N CH₂, J=14 Hz), 5.3 (d, 1H,imidazole NCH₂, J=14 Hz), 7.7 (s, 1H, imidazole NCHC), 8.1 (s, 1H,imidazole NCHC), 18.1 (br, O . . . H . . . O).

Example 6b

⁹⁹ Tc!Oxo6-hydroxy-3,3,9,9-tetramethyl-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioximato!-(3-)-N,N',N",N"'!technetium (V)

To a stirring solution of N(butyl)₄ !TcOCl₄ ⁻ (57.5 mg, 0.115 mmol) wasadded 1 mL of methanol and 150 μL of neat ethylene glycol, followed by1.5 mL of 0.75M sodium acetate in methanol. Addition of the ligand ofExample 4 (60 mg, 0.13 mmol) caused the purple solution to turn deepyellow-brown. After 5 minutes, the solvent was removed by rotaryevaporation to give a yellow-brown oil. The oil was loaded onto a 1.5×6cm silica gel column that was eluted with methylene chloride until themajor product was well separated from impurity bands at the head of thecolumn. The head of the column was removed (and discarded) and theproduct (as a very broad band) was eluted from the column with 10%methanol/90% methylene chloride. Solvent was removed and the product wasredissolved in minimal methylene chloride, washed with saturated sodiumchloride, dried over sodium sulfate and rechromatographed using 1:1ACN:CH₂ Cl₂ as the eluant. Solvent was evaporated to yield an orangeoil, which was triturated with hexanes until the product solidified. Thesolid was isolated by suction filtration, rinsed with hexanes and driedin vacuo overnight. The yield of pure title complex was 8.4 mg. M.S.:(M+H)⁺ =512, (M--H)⁻ =510. IR(KBr): 922 cm⁻¹, Tc=O.

Example 7

Preparation of ^(99m) Tc Complexes

The following general method was used to produce the ^(99m) Tc complexesof the ligands given in Examples 1-5.

Ligand (2.5 mg) was dissolved in 0.9% saline (2 mL) and 0.1M sodiumhydrogen carbonate buffer (0.5 mL) in a 10 mL glass vial. Eluate from a⁹⁹ Mo/^(99m) Tc generator (0.4 mL) was added. The vial was sealed, and asaturated solution of stannous tartrate in saline (50 μL) was added tothe vial. The vial was shaken to mix the reagents, and allowed to standat room temperature for 10 minutes.

When required, the ^(99m) Tc complex was separated from the other kitcomponents by an isolation procedure involving PRP-1 resin (as describedby S. Jurisson et al., "Chloro→Hydroxy Substitution on Technetium BATOTcCl(dioxime)₃ BR! Complexes", Nuc. Med. Biol, 18(7), 735-744 (1991).This provided the complex in ethanolic solution. The ethanol fractionwas blown dryness under nitrogen gas and redissolved in normal saline.

The radiochemical purity of the ^(99m) Tc complexes were determined byHPLC and/or TLC. HPLC analyses were conducted on a 5μ 15 cm PRP-1 columnwith 65/35 ACN/0.1M NH₄ OAc pH 4.6 as eluent at a flow rate of 1mL/min., and a radiometric detector connected to an integrator. TLCanalyses were conducted on two 20 cm SAF Instant thin layerchromatography (ITLC™) strips. 5 μL samples were applied to the originof these strips. One strip was developed with saline, and one withmethylethyl ketone (MEK). After development, strips were cut 1 cm abovethe origin, and each section was counted. The % RCP was determined as:%RCP=% on upper segment of MEK strip˜% on upper segment on saline strip.The RCP of ^(99m) Tc complexes was generally >92%.

Example 7a

^(99m) Tc! Oxo3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioximato!-(3-)-N,N', N", N"'!technetium(V) by ligand exchange from^(99m) Tc-tartrate.

To 0.5 mL of an 0.1M solution of disodium tartrate in water was added0.5 mL of physiological saline. The mixture was dispensed into acrimp-sealed vial and purged with nitrogen to remove oxygen. To this wasadded 5 μL of a freshly prepared solution of stannous chloride (2 mg/mLin degassed 1N HCl), followed by 1 mL of ^(99m) TcO₄ ⁻ eluted from a ⁹⁹Mo/^(99m) Tc generator. After 10 minutes at room temperature, theresulting Tc-tartrate complex was added to another vial that contained1.75 mg of the nitroimidazole ligand of Example 1. After 10 minutes atroom temperature, the radiochemical purity of the title ^(99m) Tc2-nitroimidazole complex was 92%, as determined by high pressure liquidchromatography conducted on a 10 micron, 15 cm PRP-1 reverse phasecolumn that was eluted with 65/35 acetonitrile/0.1M NH₄ OAc (pH 4.6) ata flow of 2 mL/minute. The complex thus prepared had a retention timethat was identical to that of an authentic sample of the ⁹⁹ Tc complexof Example 6.

Example 7b

^(99m) Tc!Oxo3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioximato!-(3-)-N,N',N",N"'!technetium(V) by ligand exchange from ^(99m)Tc-citrate

^(99m) TcO₄ ⁻ (1 ml, ˜30 mCi) in saline was added to a vial containingtrisodium citrate (0.05M) in saline (1 ml, pH adjusted to 6.1), 5 μl ofstannous chloride solution (2.2 mg/ml) in 0.1M hydrochloric acid wasadded, and the solution was allowed to stand for 10 minutes to completethe formation of ^(99m) Tc-citrate. This solution (pH 5.8) was added toa second vial containing 2 mg of the ligand described in Example 1, andthe reaction mixture was allowed to react for 20 minutes at roomtemperature. The final pH was 7.1. The radiochemical purity (determinedby HPLC, as described in Example 7A) was >94%, and the radiochemicalpurity remained at this level for >1 hour.

Example 7c

^(99m) Tc!Oxo4,7-Diaza-2,9-dimercapto-2,9-dimethyl-4-(2-(2-nitro-1H-imidazo-1-yl)ethyl)decane!-(3-)-N,N',S,S'!technetium(V)

Dithiothreitol (16.3 mg, 106 μmoles) was added to a solution of5,8-diaza-1,2-dithia-5-(2-(2-nitro-1H-imidazo-1-yl)ethyl)-3,3,10,10-tetramethylcyclodecane(6.83 mg, 18.3 μmoles, prepared as described in Example 5b) dissolved in1.0 ml methanol, and the solution was stirred at room temperature for 24hours. The volume of the reaction solution was reduced under argon to<0.25 ml and 1.25 ml pH 2.9 HBr/saline was added. The aqueous solutionwas extracted several times with diethyl ether to isolate the dithiolfrom unreacted disulfide. The ether layers were combined, blown todryness under argon, and the residue was dissolved in pH 1.6 HBr/saline.This solution was washed with diethyl ether (to remove dithiothreitol),and the pH adjusted with sodium hydroxide to 6.2 to give4,7-diaza-2,9-dimercapto-2,9-dimethyl-4-(2-(2-nitro-1H-imidazo-1-yl)ethyl)decane,which was used without further purification.

^(99m) Tc-glucoheptonate was prepared by adding ^(99m) TcO₄ ⁻ (0.3 ml,39.2 mCi) to a solution containing sodium glucoheptonate (0.5 ml of 2.42mg/ml solution in saline) and sodium acetate (0.5 ml of 0.1M; pH 7.03),followed by stannous chloride (25 μl of 5.51 mg/ml solution, 0.725μmoles, in 0.1M HCl). After standing at room temperature for 30 minutes,0.9 mL of this solution was added to a solution of4,7=diaza-2,9-dimercapto-2,9-dimethyl-4-(2-(2-nitro-1H-imidazo-1-yl)ethyl)decanein saline. The mixture was allowed to stand at ambient temperature for30 minutes, then heated to 70° C. HPLC analysis indicated two majorproducts, presumed to be syn- and anti-isomeric complexes, as found withother N-substituted-DADT complexes (e.g., L. A. Epps, H. D. Burns, S. Z.Lever, H. W. Goldfarb, H. N. Wagner, "Brain Imaging Agents: Synthesisand Characterization of (N-piperidinyl Hexamethyl Diaminodithiolate) oxoTechnetium(V) Complexes", Int. J. Appl. Radiat. Isotop, 1987,38:661-664; A. Mahmood, W. A. Halpin, K. E. Baidoo, D. A. Sweigart, S.Z. Lever, "Structure of a Neutral N-alkylated Diaminedithiol (dadt)Tc-99(V) Complex Syn TcO(NEt-tmdadt )!Tc-99", Acta Crystallogr., Sect.C: Cryst. Struct. Common., 1991, 47:254-257).

Example 8

Determination of Reduction Potential

The reduction potentials of misonidazole, ⁹⁹ TcO(PnAO), and ⁹⁹Tc-hypoxia-localizing tracers were determined by cyclic voltammetry(C.V.) in dimethylformamide. C.V. experiments employed a PrincetonApplied Research (P.A.R.) Model 174A Polarographic Analyzer with a Model303 Static Mercury Drop Electrode and were recorded on a Model RE0074X-Y Recorder. The reference electrode was Ag/AgNO₃ with an acetonitrilefilling solution saturated with LiCl. The counter electrode was aplatinum wire. Voltammograms at mercury were determined at scan rates of50, 100, 200, and 500 mV/s.

The solutions used in C.V. studies contained test sample at aconcentration of 0.2-0.7 mM and tetrabutylammonium tetrafluoroborate(Bu₄ NHF₄) or tetrabutylammonium hexafluorophosphate (Bu₄ NPF₆)supporting electrolyte at a concentration of 0.1M. The solution wasdeoxygenated by bubbling solvent-saturated nitrogen or argon through thesolution for 15 minutes. Variations in the reference potential wereaccounted for by determining the C.V. of a Ru(acac)₃ standard on a dailybasis. All measured potentials were corrected according to an absolutepeak reduction potential for Ru(acac)₃ of -1.210 V vs. Ag/AgNO₃ at Hg.The results are shown in the table below:

    ______________________________________                                        Compound name/                                                                Example number  E.sub.pc (V)                                                                          Reduction process                                     ______________________________________                                        Metronidazole   -1.62   reversible                                            Misonidazole    -1.49   reversible                                            .sup.99 TcO(PnAO)                                                                             -2.15   irreversible                                          Compound from Ex. 1                                                                           -1.52   reversible                                            Compound from Ex. 6                                                                           -1.49   reversible                                            and             -1.99   irreversible                                          ______________________________________                                    

These results demonstrate that both the ligands of this invention andthe technetium complexes thereof are reduced electrochemically atpotentials that are similar to that of the bioreducible 2-nitroimidazolecompound misonidazole, and are thus expected to undergo bioreduction invivo. In contrast, electrochemical reduction of the non-nitroimidazolecontrol Tc(V) Oxo3,3,9,9-tetramethyl-4-8-diazaundecane-2,10-dione-dioxime (TcO(PnAO)prepared by the method of Jurisson et al., Inorg. Chem., 1986, 25, 543)occurred at a potential that was far more negative than that of thefirst reduction wave observed for the compound of Example 6.

Example 8a

Demonstration of Efficacy: Reduction of the Tc Nitroimidazole Complexesby Xanthine Oxidase

The enzyme xanthine oxidase (in the presence of xanthine orhypoxanthine) is known to reduce the nitro group of suchnitroimidazole-containing compounds as misonidazole and metronidazole(see for example P. D. Josephy, B. Palcic and L. D. Skarsgard,"Reduction of Misonidazole and its Derivatives by Xanthine Oxidase",Biochem. Pharmacol., 1981, 30, 849), and it has been postulated thatsuch nitro reduction under anaerobic conditions is responsible for theselective trapping of these compounds in hypoxic tissue. Thus, atechnetium or rhenium containing nitroimidazole complex should becapable of being reduced by xanthine oxidase under anaerobic conditionsin the presence of hypoxanthine. The results from the enzyme assay belowdemonstrate that the Tc-nitroimidazole complexes of this invention arerecognized as suitable substrates by xanthine oxidase.

To a 2.5 mL quartz cuvette was added 0.25 micromoles of the ⁹⁹Tc-nitroimidazole complex of Example 6 or 6b in 125 μL ofdimethylformamide, 1 mL of 0.01M hypoxanthine in pH 7.4 sodium phosphatebuffer (0.1M), and 0.875 mL of 0.1M sodium phosphate buffer (pH 7.4)that contained 20 mg/L of disodium ethylenediamine tetraacetic acid(EDTA). The cuvette was sealed with a rubber septum, and purged withargon for 15 minutes to remove oxygen. To this was added 1.25 units ofthe enzyme xanthine oxidase (Boeringer) in 0.5 mL of deoxygenated pH 7.4phosphate buffer. The cuvette was inverted to mix, and the UV/visiblespectrum of the solution was recorded from 280 to 600 nm at 15 minuteintervals.

The absorbance peak at approximately 320 nm, which is characteristic ofthe nitroimidazole functionality, decreased in intensity. It is believedthat the disappearance of this nitro absorbance is due to reduction ofthe nitro group by xanthine oxidase. In a control reaction thatcontained no enzyme, no spectral changes were observed over a period of7 hours.

In a parallel control reaction, the reagents above were mixed in thesame fashion, but the ⁹⁹ Tc complex of3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione dioxime (prepared bythe method of Jurisson et al., Inorg. Chem., 1986, 25, 543) wassubstituted for the technetium complexes of Examples 6 or 6b. In thisreaction, which did not contain a bioreducible nitroimidazolefunctionality, no spectral changes were observed over a period of 7hours.

Example 9 Demonstration of the Ability to Cross Endothelial Monolayers

Bovine brain microvessel endothelial cells were isolated using amodification of the Audus-Borchardt method (K. L. Audus et al., Ann. NewYork Acad. Sci., 1988; 9-18). The measurements of bovine brainmicrovessel endothelial permeability in vitro were adapted from modelsby Audus and Borchardt (K. L. Audus et al., J. Neurochem., 1986;47:484-488 and M. V. Shah et al., Pharm. Res. 1989; 6:624-627) and W. M.Pardridge et al. (J. Pharmacol. Exptl. Therap., 1990; 253:884-891)except that Anocell inserts were used in place of Transwells containingpolycarbonate filters, or polycarbonate filters placed into aside-by-side apparatus. The use of electrical resistance as anindication of tight junction formation (P. Artursson et al., J. Pharm.Sci., 1990; 79:595-600 and S. G. Milton et al., J. Cell. Physiol., 1990;144:498-504) was applied by using the Millicell-ERS resistance systemfrom Millipore. An asymptotic level of high electrical resistance (˜600Ohms-cm²) at morpho-logical confluence indicated tight junctionformation. Only those wells with resistance ≧500 Ohms-cm² were used.Further modifications of the Audus-Borchardt method were the use ofDMEM/F-12 media with 10% plasma derived horse serum as the experimentalmedium inside the Anocell insert and in the outer well.

A study of the permeability of a single test compound utilized 12Anocell inserts:

4 wells containing monolayers, 0.4 mL of media with 10% plasma-derivedhorse serum, 5 μCi of ³ H-water, 2 μCi of ¹⁴ C-sucrose, and 20 μCi ofthe Tc-99m complex;

4 wells containing the same as above, but without the monolayers;

and 4 wells containing the same but with neither monolayers nor the 10%plasma-derived horse serum.

This system was placed into a 37° C., 5% CO₂ incubator which contains anorbiting tissue culture plate shaker for agitation and 10 μL samples,from both inside (donor) and outside (acceptor) compartments, were takensimultaneously from a set of 4 Anocell inserts. These samples werecounted first in a Gamma counter then, after 72 hours, in ascintillation counter with dual channel capabilities. The fraction ofradio activity transported from the donor to the acceptor wells at eachtime point over the first 10 minutes of the study was calculated. Theaverage percent of radioactivity transported was plotted vs. time andthe slope was estimated by linear regression analysis. The slope of theclearance curve with filter alone is equal to PS_(f), wherePS=permeability surface area product. The slope of The clearance curveof the wells containing the filter plus endothelial cells was denotedPS_(m). The slope of the clearance curve was linear up to 10 minutes forall agents tested. The corrected PS value for The endothelial monolayer,called PS_(e), was computed as follows (according to Pardridge et al.,J. Pharmacol. Exptl. Therap., 1990, 253, 884-891): ##EQU1## Thepermeability index (P_(i)) is calculated using the PS_(e) for each agentas follows: ##EQU2## The following table provides The determined P_(i)values for several of the compounds examined:

    ______________________________________                                        Compound name/Example number                                                                        P.sub.i                                                 ______________________________________                                        .sup.99m Tc-PnAO (1)  64.4                                                    .sup.99m Tc-HM-BAT (2)                                                                              44.3                                                    .sup.99m Tc-TMR (3)   50.3                                                    .sup.99m TcCl(DMG).sub.3 2MP (4)                                                                    -9.3                                                    .sup.99m Tc complex from ligand in Ex. 1                                                            63.2                                                    .sup.99m Tc complex from ligand in Ex. 5                                                            0.2                                                     .sup.99m Tc complex from ligand in Ex. 2                                                            15.2                                                    .sup.99m Tc complex from ligand in Ex. 4                                                            1.8                                                     .sup.99m TcCl(DMG).sub.3 BBNO.sub.2 (5)                                                             4.5                                                     ______________________________________                                         (1) W. A. Volkert, T. J. Hoffman, S. M. Seger, D. E. Troutner, R. A.          Holmes, "Tc99m Propylene Amine Oxime (Tc99m PnAO); A Potential Brain          Radiopharmaceutical", Eur. J. Nucl. Med. 1984, 9:511-516.                     (2) H. F. Kung, M. Molnar, J. Billings, R. Wicks, M. Blau, "Synthesis and     Biodistribution of Neutral LipidSoluble Tc99m Complexes that Cross the        BloodBrain-Barrier", J. Nucl. Med., 1984, 25:326-332.                         (3) R. H. Mach, H. F. Kung, YZ, Guc, CC Yu, V. Subramanyam, J. C.             Calabrese, "Synthesis, Characterization and Biodistribution of Neutral an     LipidSoluble .sup.99m TcPAT-HM and .sup.99m TcTMR for Brain Imaging",         Nucl. Med. Biol., 1989, 16:829-837.                                           (4) E. N. Treher, L. C. Francesconi, J. Z. Gougoutas, M. F. Malley, A. D.     Nunn, "Monocapped Tris(dioxime) Complexes of Technetium(III): Synthesis       and Structural Characterization of TcX(dioxime).sub.3 BR (X = Cl, Br;         dioxime = dimethylglyoxime, cyclohexanedione dioxime; R = CH.sub.3,           C.sub.4 H.sub.9), Inorg. Chem., 1989, 28:3411-3416.                           (5) K. E. Linder, S. Jurisson, A. D. Nunn, "Boronic Acid Adducts of           Technetium99m Dioxime Complexes and Rhenium Dioxime Complexes Containing      Biochemically Active Group, European Patent No. 411,491; 1991.           

Example 9a The Biodistribution of ^(99m) Tc-Complexes in Normal(Normoxic) Sprague-Dawley Rats

The biodistribution of ^(99m) Tc-complexes was determined to demonstratedelivery of the radiotracers to the target organs, and the clearance ofradioactivity from normoxic tissue in the target area and nearbytissues.

Twelve Sprague-Dawley rats were anesthetized with Nembutal (50 mg/kg)and injected with 0.1 mL (20 μCi) of radioactivity via the jugular vein.At 1 minute, 5 minutes and 60 minutes after administration of theradiotracers (n=4 for all time points), the animals were sacrificed byexsanguination, and the target tissues removed, weighed and assayed forradioactivity. The rats were allowed to respire room air throughout thecourse of the study.

The results are shown in the following tables:

                  TABLE 1                                                         ______________________________________                                               Percent ID/g for the                                                          .sup.99m Tc-complex of the ligand in Example 1                                1 Min.    5 Min.      60 Min.                                          Tissue   MEAN    SEM     MEAN  SEM   MEAN  SEM                                ______________________________________                                        Brain    0.30    0.03    0.11  0.01  0.02  0.00                               Blood    0.47    0.06    0.36  0.01  0.28  0.02                               Heart    1.32    0.19    0.34  0.03  0.09  0.01                               Lungs    0.71    0.09    0.39  0.03  0.16  0.01                               Kidneys  2.59    0.27    1.15  0.12  0.45  0.04                               Liver    2.06    0.25    3.63  0.18  2.45  0.18                               Muscle   0.25    0.07    0.14  0.02  0.05  0.02                               Bone     0.37    0.02    0.23  0.01  0.11  0.05                               Stomach  0.67    0.10    0.56  0.03  1.80  0.34                               Thyroid  0.65    0.10    0.77  0.28  0.15  0.02                               Thymus   0.55    0.06    0.25  0.06  0.05  0.00                               Upper Intestine                                                                        1.13    0.05    0.98  0.09  4.60  0.22                               Lower Intestine                                                                        0.50    0.06    0.40  0.03  0.36  0.04                               Bladder  0.15    0.02    0.68  0.12  2.84  0.41                               Spleen   0.84    0.02    0.45  0.02  0.23  0.03                               ______________________________________                                         (SEM = standard error of the mean)                                       

                  TABLE 2                                                         ______________________________________                                               Percent ID/g for the                                                          .sup.99m Tc-complex of the ligand in Example 4                                1 Min.    5 Min.      60 Min.                                          Tissue   MEAN    SEM     MEAN  SEM   MEAN  SEM                                ______________________________________                                        Brain    0.03    0.00    0.01  0.00  0.00  0.00                               Blood    0.62    0.07    0.29  0.01  0.09  0.01                               Heart    1.02    0.08    0.22  0.01  0.05  0.00                               Lungs    0.63    0.03    0.25  0.01  0.05  0.01                               Kidneys  2.53    0.22    0.94  0.04  0.70  0.04                               Liver    2.19    0.14    2.69  0.19  1.09  0.05                               Muscle   0.19    0.05    0.16  0.02  0.04  0.00                               Bone     0.34    0.00    0.16  0.01  0.03  0.00                               Stomach  0.30    0.06    0.25  0.04  0.23  0.03                               Thyroid  0.61    0.05    0.37  0.02  0.08  0.01                               Thymus   7.01    0.26    0.20  0.01  0.04  0.00                               Upper Intestine                                                                        0.72    0.06    1.18  0.21  5.36  0.14                               Lower Intestine                                                                        0.29    0.04    0.45  0.16  0.39  0.01                               Bladder  0.09    0.03    2.59  0.75  6.40  1.12                               Spleen   0.94    0.10    0.41  0.02  0.09  0.02                               ______________________________________                                         (SEM = standard error of the mean)                                       

Example 10 Demonstration of Efficacy in a Rabbit Focal MyocardialIschemia Model

A model of focal myocardial ischemia in the rabbit was developed, usingpermanent ligation of the left anterior descending (LAD) coronaryartery. The model consisted of two studies. In the first, relativeregional myocardial blood flow (MBF) and relative regional myocardialrate of glucose metabolism (MMR_(g1)) were determined by autoradiographyusing a double-label study with the flow tracer ^(99m) TcCl(CDO)₃ MeB(R. K. Narra et al., J. Nucl. Med., 1989; 30:1830-1837) and ¹⁴ C-deoxyglucose for MMR_(g1) (L. Sokoloff et al., J. Neuro-chem., 1977, 28,897-916). ¹⁴ C-deoxyglucose and the ^(99m) Tc-hypoxia-localizing tracerwere administered to a second group of rabbits with LAD coronary arteryocclusion.

After the surgical preparation and 20 minutes of LAD occlusion, ¹⁴C-deoxyglucose (130-150 μCi) was injected as an intravenous bolus, andtimed arterial blood samples were obtained. Twenty-five minutes later,^(99m) TcCl(CDO)₃ MeB (10-12 mCi) was administered intravenously. Fiveminutes later, the rabbits were sacrificed by intravenous injection ofNembutal and potassium chloride. The heart was excised, frozen in liquidFreon-22, and 20 μm coronal sections obtained with a Microtome.Autoradiographs were obtained on all sections. For the first exposure(˜14 hours duration) of Kodak XAR film, extra heavy duty aluminum foilwas interposed between the tissue and film to block completely theradiation emanating from ¹⁴ C to obtain the MBF information derived from^(99m) TcCl-(CDO)₃ MeB alone. After three days (to allow for decay of^(99m) Tc), a second autoradiograph was obtained without foil. Thesecond exposure lasted 6-8 days, and provided an image of the regionaldistribution of ¹⁴ C-deoxyglucose. These images established that thereis a zone of increased glycolysis bordering on the ischemic territory.This region of increased glycolysis, induced by reduced tissue pO₂,marks the hypoxic ischemic border zone.

In a second group of animals, the protocol was similar, except that the^(99m) Tc complex of the ligand described in Example 1 was co-injectedwith ¹⁴ C-deoxyglucose, and the animal sacrificed 30 minutes later.

Autoradiography revealed an isomorphic relationship between the regionalmyocardial distribution pattern of this complex and that for ¹⁴C-deoxyglucose. Both tracers displayed high uptake in the ischemicborder zone, with low levels of radioactivity in the regions of normalperfusion, and virtually no radioactivity in the region of no flow. Themicroregional distribution of both the tracers was virtually identical.By comparison, the ischemic zone in the study with the ^(99m) Tc-flowtracer showed little accumulation of radioactivity, while regions ofnormal perfusion displayed high levels of radioactivity.

The ^(99m) Tc-complex of3,3,6,9,9-pentamethyl-4,8-diazaundecane-2,10-dione dioxime was examinedin this model as an example of a ^(99m) Tc-PnAO-complex which does notpossess a hypoxia-localizing functionality. The autoradiograms obtainedwith this tracer showed no differentiation of ischemic and non-ischemicregions indicating that a hypoxia localizing moiety such as2-nitroimidazole is essential for specific localization of thesecomplexes within hypoxic regions.

In a separate experiment using the rabbit LAD occlusion model and thedouble-label auto radiography procedures described above, theperformance of the ^(99m) Tc complex of the ligand described in Example1 was compared to that of ¹⁴ C-misonidazole. The microregionaldistribution of both agents was virtually identical and was similar tothat found previously for ¹⁴ C-deoxyglucose: high uptake in the hypoxicborder zone of the ischemic territory and low uptake in normoxic regionsand in the center of the ischemic territory where flow is limiting.

Example 10a Demonstration of Efficacy in a Rat Focal Cerebral IschemiaModel

A model of focal cerebral ischemia involving tandem occlusion of theinternal carotid artery and the ipsilateral middle cerebral artery (MCA)in spontaneously hypertensive rats (SHR) was characterized using thedouble-label autoradiography procedure described in Example 10. In thiscase, ^(99m) TcCl(DMG)₃ 2MP (Narra et al., J. Nucl. Med., 1990, 31(8),1370-1377) was used as the indicator for cerebral blood flow (CBF) and,as before, ¹⁴ C-deoxyglucose was used to demonstrate areas of increasedglycolysis indicative of tissue hypoxia. Following surgery, which wasperformed under Halothane anesthesia, the rats were allowed to recoverand one hour after the MCA occlusion, ¹⁴ C-deoxyglucose was injected asan IV bolus and timed arterial samples were obtained. Twenty-fiveminutes after ¹⁴ C-deoxyglucose injection, ^(99m) TcCl(DMG)₃ 2MP wasinjected as an IV bolus and the rat was sacrificed 15 seconds later. Thebrain was rapidly removed and sections and autoradiograms were obtainedas described in Example 10. As found in Example 10 for the rabbit LADocclusion model, the ischemic territory was bordered by a rim of tissuein which glycolysis was elevated. Unlike the previously cited example ofmyocardial ischemia, the hypoxic region in the brain did not have asgreat an increase in glycolysis compared to normoxic regions because thebrain uses glucose as the preferred substrate for oxidation in normoxictissue. Nevertheless, it was clear that the ischemic region issurrounded by a border zone of increased glycolysis. In a second seriesof experiments, the ^(99m) Tc complex of the ligand described in Example1 and ¹⁴ C-deoxyglucose were co-injected 1 hour or five days after MCAocclusion. Autoradiograms were obtained as described above and revealed,for both time points, that both agents displayed an increased uptake inthe hypoxic border zone relative to surrounding normoxic tissue.Moreover computer assisted image analysis showed that, in the case of^(99m) Tc complex of the ligand described in Example 1, thehypoxic-normoxic optical density ratio was 7:1. These findingsdemonstrate the efficacy of the ^(99m) Tc complex of the liganddescribed in Example 1 for both acute and chronic episodes of focalcerebral ischemia.

Example 11 Demonstration of Efficacy: Isolated Perfused Heart Studies

Hearts were excised from male Sprague Dawley rats (275-325 g) and wereperfused retrogradely using the Langendorff method (O. Langendorff,Pfleugers Arch. ges. Physiol, 61, 291, 1985) with modificationsdescribed previously (W. Rumsey, D. F. Wilson and M. Erecinska, Am. J.Physiol., 253 (Heart Circ. Physiol. 22): H1098, 1987) in the isolatedstate at 37° C. with Krebs-Henseleit buffer. The perfusate contained inmM! NaCl 118!, KCl 4.7!, CaCl₂ 1.8!, Na₂ EDTA 0.5!, KH₂ PO₄ 1.2!, MgSO₄1.2!, NaHCO₃ 25!, glucose 11!, pyruvate 0.2!, and insulin (12 IU/L) andwas equilibrated with O₂ :CO₂ (95:5) (global normoxia) or N₂ :CO₂ (95:5)(global hypoxia). The hearts were paced continuously at 5 Hz. Perfusionpressure was maintained at 72 cm H₂ O for 20 minutes in order to allowthe hearts to adjust to the isolated state. After this initialadjustment period, perfusate flow was maintained constant a levelsimilar to that obtained at the end of the adjustment period, i.e., 7-8mL/min/g wet weight, using a peristaltic pump.

For determination of oxygen consumption, a cannula was placed in theright ventricle via the pulmonary artery. A pump removed a smallfraction of the coronary effluent at 1 mL/min., and its oxygenconcentration was monitored continuously by an in-line Clark-typeelectrode. In the normoxic state, the influent oxygen concentration wasmaintained at 956 μM. Coronary flow was measured by collecting theeffluent from the right and left pulmonary arteries in a 10 mL graduatedcylinder. Oxygen consumption was calculated from the product of theinfluent-effluent oxygen concentration difference and the coronary flow.During perfusion with hypoxic medium, only the effluent oxygenconcentration was recorded. Typically, effluent oxygen concentration was505 μM in the normoxic studies and 17 μM in the hypoxic studies.

The heart was perfused with either normoxic or hypoxic medium for 30minutes prior to the administration of the test compound. The Tc-99mtracer was administered over 20 minutes by infusion into the perfusate,and radioactivity in the perfused heart was detected by a collimatedcrystal positioned 3-4 cm from the right ventricle and perpendicular tothe vertical axis of the heart. The radioactivity remaining in the heartat 40 minutes after the end of the infusion period was divided by thepeak level of radioactivity to give a measure of retention. Results(n=4) are shown in the table, below:

    ______________________________________                                        % Retention of Tracer in the Isolated Perfused Rat Heart                                        Normoxia                                                                              Hypoxia                                             ______________________________________                                        Tc-99m complex of ligand in Ex. 1                                                                 33.5 ± 2.5                                                                           65.3 ± 3                                      Tc-99m!TcCl(CDO).sub.3 MeB**                                                                      71.3 ± 5.5*                                                                         63.3 ± 3.7                                    Tc-99m!TcCl(DMG).sub.3 2MP**                                                                     68.5 ± 0.5                                                                           48.7 ± 1.3                                   ______________________________________                                         *n = 3                                                                        **Prior Art Boronic Acid Adducts (U.S. Pat. No. 4,705,849)               

The Tc-99m complex of ligand 1 demonstrates greater retention in thehypoxic heart, compared to the normoxic heart. By comparison, the flowtracers TcCl(DMG)₃ 2MP and TcCl(CDO)₃ MeB do show an increase inretention under hypoxic conditions compared to normoxia.

Example 12 Demonstration of Efficacy: Isolated Cardiac Myocyte studies

Calcium-tolerant ventricular myocytes were isolated from hearts of maleSprague Dawley rats (200 g) according to the procedure of Wittenberg andRobinson (B. A. Wittenberg and T. F. Robinson, Cell Tissue Res., 216:231, 1981) with modifications described previously (W. Rumsey, C.Schlosser, E. M. Nuutinen, M. Robiolio and D. F. Wilson, J. Biol. Chem.,265 (26): 15392, 1990). Cells were used immediately followingmorphological analysis (using a hemocytometer) of viability and weremaintained at 37° C. during the experiments. The number of quiescent,rod shaped cells ranged from 70-90% within a total population of 5-9×10⁶cells.

The isolated myocytes were maintained in either a normoxic, hypoxic oranoxic state. Hypoxia was induced by providing an atmosphere of argonatop of the cells and sealing the flask during the incubation period.Glucose oxidase plus catalase (5/5 mg) was added to argon treated cellsto provide anoxia. Cells were suspended (6.5-7.5×10⁴ cells/ml) inisolation media and aliquots were added to incubation vials maintainedat 37° C.

After incubation with a test compound, myocytes were deproteinated with1% ice-cold perchloric acid and centrifuged at 12,000 rpm for 30 sec.The supernatent was separated from the pellet and each counted using aLKB 1282 gamma counter. Alternatively, myocytes were separated from thesuspending media by passing the cells through 99% dibutyl phthalate bycentrifugation at 12,000 rpm for 30 sec. The three phases were separatedand counted as described above. Results for the Tc-99m complex of thecompound in Example 1 are given below:

    ______________________________________                                        Condition        PCA Pellet                                                                              Cell Pellet                                                                            Oil                                       ______________________________________                                        Normoxia (n = 5) 40.5 ± 2.7                                                                           23.2 ± 3.7                                                                          22.8 ± 2.2                             Hypoxia (Argon) (n = 4)                                                                        48.5 ± 3.4                                                                           36.9 ± 8.8                                                                          20.1 ± 5.3                             Anoxia (Glucose Oxidase) (n = 4)                                                               55.5 ± 5.8                                                                           48.8 ± 4.2                                                                           9.0 ± 2.6                             ______________________________________                                    

Values represent means ± S.E.M. for the number of experiments noted inthe parentheses. The values are the percent of total radioactivityrepresented by each case. PCA pellet=perchloric acid precipitated pelletwhich represents the activity associated with proteins/membranes. Cellpellet=whole cells passed through a layer of dibutyl phthalate (oil).

These data demonstrate that the Tc-99m complex of the compound inExample 1 shows retention in the sequence anoxia>hypoxia>normoxia. Sincea significant proportion of tracer was retained in normoxic myocytes, aseparate study with isolated myocytes was undertaken.

Addition of an uncoupler of β-oxidative phosphorylation, carbonylcyanide p-trifluoro methoxyphenylhydrazone (FCCP), which completelyoxidizes the mitochondrial electron transport chain (NADH/AND⁺ ratio andthe redox potential of the cells decrease) but decreases thephosphorylation potential ( ATP!/ ADP! Pi!) to levels similar to thatfound in hypoxia, had no effect on the retention of the Tc-99m complexof the compound in Example 1 in normoxic cells. Moreover, addition ofcyanide to separate cell suspensions (n=3), which inhibits electrontransport between cytochrome oxidase and oxygen (NADH/NAD⁺ ratio andredox potential increase) but also decreases the phosphorylationpotential to very low levels, had no effect on retention in normoxiccells. These results suggest that:

1) retention of the Tc-99m complex of the compound in Example 1 innormoxic cells is not dependent on the redox state of theintramitochondrial pyridine nucleotides and is most likely due to itslipophilicity or other molecular interactions affecting binding tocellular material. If the amount retained in normoxic cells wasdependent upon the redox state, retention would have been expected todecrease upon addition of FCCP.

2) Significant retention of the Tc-99m complex of the compound inExample 1 requires an oxygen-free or low oxygen milled. An increase inthe redox potential (cyanide) is not sufficient to affect the level ofretention. The latter results were confirmed using Na Amobarbital whichalso inhibits electron flux but at site I of the respiratory chain(NADH/NAD⁺ ratio increases).

3) Most importantly, by uncoupling the cells and by addition of cyanide,the energy state of the cells was reduced to levels that were likelysimilar to that obtained with oxygen 30 deprivation. Thus, any changesin cellular permeability, geometry and viability were also similar,suggesting that retention was due to reduction of the nitro moiety ofthe Tc-99m complex of the compound in Example 1 in the absence ofoxygen. These data indicate that, in hypoxia, the Tc-99m complex of thecompound in Example 1 becomes trapped within the hypoxic cells.

When cellular integrity was disrupted by freezing and thawing the cells(3×) before incubation with the Tc-99m complex of the compound inExample 1 under normoxic and hypoxic (glucose oxidase) conditions, thepercentage of activity associated with the protein/membrane fragmentswas similar (normoxic=23.3±0.8%, anoxic=25.3±2.5%, n=3). The latterindicates that an intact cell is required for trapping of the compound.

Several Tc-99m complexes were examined in isolated myocytes using theprotocol described above. The percentage of activity retained in thecell pellet was determined under anoxic and normoxic states. Results areshown below:

    ______________________________________                                                   % in Cell Pellet                                                   ______________________________________                                                     .sup.99m Tc Complex of                                                                     .sup.99m Tc Complex of                                           Compound in Ex. 1                                                                          Compound in Ex. 2                                   Normoxia     24.1 ± 2.3                                                                              23.5 ± 1.7                                       Anoxia       53.0 ± 2.2                                                                              39.9 ± 0.5                                       Anoxia/normoxia                                                                            2.3          1.7                                                 ratio                                                                                      .sup.99m Tc Complex of                                                                     .sup.99m Tc Complex of                                           Compound in Ex. 4                                                                          Compound in Ex. 5                                   Normoxia     10.9 ± 0.8                                                                              10.0 (n = 1)                                        Anoxia       48.6 ± 9.8                                                                              18.2 (n = 1)                                        anoxia/normoxia                                                                            4.4          1.8                                                 ratio                                                                                      .sup.99m Tc Complex of                                                                     .sup.99m Tc Complex of                                           6-methy1-PnAO                                                                              6-hydroxy-PnAO                                      Normoxia     17.4 (n = 1)  7.1 (n = 1)                                        Anoxia       22.7 (n = 1)  9.2 (n = 1)                                        anoxia/normoxia                                                                            1.3          1.3                                                 ratio                                                                                      .sup.99m Tc Complex of                                                                     .sup.99m Tc Complex of                                           TcCl(CDO).sub.3 MeB                                                                        TcCl(DMG).sub.3 2MP                                 Normoxia     73.6 (n = 2) 69.5 (n = 2)                                        Anoxia       83.5 (n = 2) 80.0 (n = 2)                                        anoxia/normoxia                                                                            1.1          1.2                                                 ratio                                                                         ______________________________________                                    

The ^(99m) Tc-complexes of 6-methyl PnAO(3,3,6,9,9-pentamethyl-4,8-diazaundecane-2,10-dione dioxime) and6-hydroxy PnAO(6-hydroxy3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione dioxime) and the ^(99m)Tc-complexes TcCl(CDO)₃ MeB and TcCl(DMG)₃ 2MP (U.S. Pat. No. 4,705,849)are representative neutral, lipophilic complexes which do not contain ahypoxia-localizing moiety. These data demonstrate greateranoxia/normoxia ratios for the hypoxia-localizing compounds of thisdisclosure than the complexes without a hypoxia localizing moiety.

In a separate study, the uptake of the ⁹⁹ Tc complex of the ligand ofExample 1 in isolated myocytes under normoxia, hypoxia and anoxia wascompared to ³ H-FMISO and ¹²⁵ I-iodovinyl MISO. The anoxia/normoxia andhypoxia/normoxia ratios indicate that the ^(99m) Tc complex of thecompound in Example 1 shows similar selective retention in anoxic cellsto the hypoxia-localizing compounds labeled with ³ H and ¹²⁵ I describedin the literature.

    ______________________________________                                                 .sup.99m Tc Complex of                                                                             .sup.125 I-iodovinyl                                     ligand in Ex. 1                                                                          .sup.3 H-FMISO                                                                          MISO                                            ______________________________________                                        Normoxia   18 ± 1 (3)                                                                              3 ± 1 (3)                                                                            12 ± 1 (3)                               Hypoxia    30 ± 7 (3)                                                                              5 ± 1 (3)                                                                            16 ± 3 (2)                               Anoxia     48 ± 6 (3)                                                                              8 ± 2 (3)                                                                            24 ± 3 (3)                               Hypoxia/normoxia                                                                         1.7          1.7       1.4                                         Anoxia/normoxia                                                                          2.7          2.7       2.0                                         ______________________________________                                    

Example 13 Demonstration of Efficacy: Studies in Isolated Mitochondria

Mitochondria were prepared from hearts of male Sprague Dawley rats (200g) using the isolation procedure of Fuller et al., (E. O. Fuller, D. I.Golderg, J. W. Starnes, M. Sacks, and M. J. Delavoria-Papadopoulos, Mol.Cell. Cardiol., 17:71, 1985). The heart was excised from anesthetizedrats and trimmed free of the atria and great vessels. The ventricleswere minced in ice-cold isolation medium (0.225M mannitol, 75 mMsucrose, 1.0 mM EGTA and 10 mM MOPS, pH 7.4), briefly exposed to theproteolytic enzyme preparation, Nagase (Enzyme Development Corp., NewYork, N.Y.), and homogenized with a polytron. The mitochondria wereseparated from 20 the remainder of the broken cells using densitygradient centrifugation.

Three nitroimidazole compounds were incubated at 37° C. for 60 minutesin isolated mitochodria. Hypoxia and anoxia were induced as outlined inExample 12. The percentage of radioactivity associated with themitochondria are shown in the following table:

    ______________________________________                                                   .sup.99m Tc Complex of                                                                   .sup.99m Tc Complex of                                             Ligand in Ex. 1                                                                          Ligand in Ex. 4                                         ______________________________________                                        Normoxia     27.8 ± 1.2                                                                              15.7 ± 0.4                                       Hypoxia      39.2 ± 1.0                                                                              47.7 ± 3.7                                       Hypoxia/Normoxia                                                                            1.4 ± 0.1                                                                               3.1 ± 0.3                                       ______________________________________                                    

Values are given in percent of total radioactivity within an aliquot andrepresent means ±S.E.M. Compounds were tested using the same preparationof mitochondria.

These data indicate that mitochondria may have a role in the selectiveretention of these radiotracers under hypoxic and normoxic conditions.

Example 14 Synthesis of3,3,6,6,9,9-Hexamethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. N-(3-Amino-2,2-dimethylpropyl)-1-amino-1,1-dimethyl-2-butanone oxime

To a solution of 2,2-dimethyl-1,3-propane diamine (69 g. 0.75 mole) indry methanol (100 ml), 3-chloro-3-methyl-2-nitrosobutane (20.55 g, 0.15mole, Example 1) was added in portions at 0° C. over a period of 2hours. The reaction mixture was then stirred at room temperature for 20hours. The solvent was removed under reduced pressure to give a paste.Water (50 mL) was added, and the solution was cooled in an ice bath. Thesolution was filtered and the filtrate was adjusted to pH 10-11 by theaddition of sodium hydroxide. The solution was cooled again andfiltered. The filtrate was concentrated under reduced pressure to apaste and then extracted with ether repeatedly (10×50 mL). The combinedether solution was concentrated to give an oil which was recrystallizedtwice from petroleum ether to yield the title A compound as a colorlesscrystalline solid (20.0 g), m.p. 58°-60° C. ¹ H NMR CDCl₃ !: δ 0.85 (s,6H, C--Me₂), 1.28 (s, 6H, N--CMe₂), 1.9 (s, 3H, N═CMe), 2.3 (s, 2H,NCH₂) and 2.6 (s, 2H, N--CH₂).

B.3,3,6,6,9,9-Hexamethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A solution of the the title A compound (0.8 g, 4 mmol) was treated withdiisopropylethylamine (0.39 g, 3 mmol) in dichloromethane (5 mL) andstirred. 3-Chloro-3-methyl-1-(2-nitro-1H-imidazo-1-yl)-2-nitrosobutane(0.783 g, 3 mmol, Example 1(B)) was added and the reaction mixture wasstirred at room temperature for 48 hours. All volatile material wasremoved under reduced pressure, and the resultant paste was dissolveddichloromethane (2 mL). This solution was loaded onto a flash silica gelcolumn. The column was slowly eluted with 0-5% methanol in dichloromethane until all of the product had eluted. The crude product waspurified by chromatography twice more to give a pale yellow product with˜97% purity by HPLC analysis. The product was dried under vacuum at roomtemperature for 24 hours to give 0.12 g of the title compound, m.p:--thesolid becomes a glass at 80°-83° C. and melts at 118°-120° C. withdecomposition.

¹ H NMR CDCl₃ !: δ0.8 (s, 6H, CMe₂), 1.2 (s, 12 H, N--CMe₂), 1.9 (s, 3H,N═CMe), 2.2 (2s d, 4H, N--CH₂), 5.4 (s, 2H, imidazole CH₂), 7.1 (s, 1H,imidH) and 7.15 (s, 1H, imidH). M.S. M+H!⁺ 412. Analysis calc'd for C₁₈H₃₃ N₇ O₄ . 0.6 THF and 0.1 H₂ O: C, 53.73; H, 8.39; N, 21.50; Found: C,53.73; H, 8.55; N, 21.28.

Example 15 Synthesis of6,6-Diethyl-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. N-(2-Aminomethyl-2-ethylbutyl)-1-amino-1,1-dimethyl-2-butanone oxime

3-Chloro-3-methyl-2-nitrosobutane (4.59 g, 0.034 mol) was addedportionwise to a cooled (0° C.) solution of5,5-diethyl-1,3-diaminopropane (8.86 g, 0.068 mol) in methanol (40 mL).After the addition, the reaction mixture was allowed to warm to roomtemperature and stirred for 48 hours. Methanol was removed on a rotaryevaporator. The residue was dissolved in dioxane-water (2:1, 300 mL) andthe solution was cooled to 0° C. Sodium carbonate (15.9 g, 0.15 mol) wasadded to this mixture followed by di-t-butyl dicarbonate (32.73 g, 0.15mol). The reaction mixture was stirred at 0° C. for 2 hours and at roomtemperature for 12 hours. Dioxane and water were removed on a rotaryevaporator and the residue was poured into water and extracted withether. The ether solution was washed with water and dried with sodiumsulfate. Ether was removed on a rotary evaporator and the residue waschromatographed over silica gel (hexane-ethyl acetate, 7:3).Di-t-Boc-5,5-diethyl-1,3-diamino propane eluted in the earlierfractions. The fractions containing the Boc derivative of the productwere collected and the solvent was evaporated to yield a thick oil whichsolidified on standing (4.2 g). This was treated with methanolic HCl (25mL) at room temperature for 30 minutes. Methanol was removed underreduced pressure and the solid obtained was neutralized with methanolicammonia to yield the title A compound as a white solid. This was usedfor the next step without further purification. ¹ H NMR (D₂ O) δ0.8 (t,6H, CH₃), 1.43 (q, 4H, CH₂), 1.52 (s, 6H, C(CH₃)₂), 1.84 (s, 3H, CH₃),2.99 (d, 4H, CH₂).

B. 6,6-Diethyl-3,3,9,9-tetramethyl-1-(2-nitro-1Himidazo-1-yl)-4,8-diazaundecane-2,10-dione dioxime

Diisopropylethylamine (0.65 g, 0.005 mol) was added to a slurry ofN-(2-aminomethyl-2-ethylbutyl)-1-amino-1,1-dimethyl-2-butanone oxime(1.15 g, 0.005 mol) and3-chloro-3-methyl-1-(2-nitro-1H-imidazo-1-yl)-2-nitrosobutane (1.23 g,0.005 mol, Example 1) in acetonitrile. The reaction mixture was stirredat room temperature for 48 hours. Acetonitrile was removed under reducedpressure and the residue was chromatographed over silica gel (methylenechloridemethanol, 95.5:0.5). Fractions containing the product werecollected and evaporated on a rotary evaporator. The resultant oil wasdissolved in a minimum amount of CHCl₃ and left in the refrigerator. Thesolid which formed was removed by filtration, and air dried (0.62 g),m.p. 124°-125° C.

Analysis calc'd for C₂₀ H₃₇ N₇ O₄ : C, 54.64; M, 8.48; N, 22.29; Found:C, 54.45; M, 8.50; N, 22.16.

Example 16 Synthesis of6,6-Diethyl-3,3,9,9-tetramethyl-1-(4-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

Diisopropylethylamine (0.65 g, 0.005 mol) was added to a slurry ofN-(2-aminomethyl-2-ethylbutyl)-1-amino-1,1-dimethyl-2-butanoneoxime(0.46 g, 0.002 mol, Example 15) and3-chloro-3-methyl-1-(4-nitro-1H-imidazo-1yl)-2-nitrosobutane (0.47 g,0.002 mol, Example 2) in acetonitrile was added and the mixture wasstirred at room temperature for 48 hours. Acetonitrile was removed underreduced pressure and the residue was chromatographed over silica gel(methylene chloride-methanol, 80:20). UV positive fractions werecollected and evaporated on a rotary evaporator. The light yellow oilobtained solidified on standing (0.52 g) . ¹ H NMR (DMSO-d₆): δ0.76 (m,6H, CH₃), 1.24 (m and S, 16H, CH₂ CH₃ and C (CH₃)₂), 1.48 (s, 3H, CH₃)1.73 and 1.85 (s, 4H, CH₂ NH), 5.02 (s, 2H, N--CH₂), 7.8 and 8.24 (s,2H, imi.H), 11.1 and 11.8(s, 2H, N--OH).

Analysis calc'd for C₂₀ H₃₇ N₇ O₄ . 2.71 H₂ O: C, 49.19; H. 8.75; N,20.08; Found: C, 49.17; H, 8.13; N, 19.72.

Example 17 Synthesis of3,3,9,9-Tetramethyl-1,11-bis2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A slurry of3-chloro-3-methyl-1-(2-nitro-1H-imidazo-1-yl)-2-nitrosobutane (0.5 g,0.002 mol, Example 1) in acetonitrile (5 mL) was maintained an 45° C.for 10 minutes. To this suspension was added a mixture of1,3-propanediamine (75 mg, 0.001 mol) and diisopropylethylamine (300 mg,0.002 mol). The stirred mixture was maintained at 45° C. for 15 min. Aclear solution was formed in 10 minutes. Acetonitrile anddiisopropylethylamine were removed on a rotary evaporator and theresidue was dissolved in water (0.5 mL) and made basic with aqueousammonia. The solution was extracted with ethyl acetate, and the ethylacetate layer was removed and dried with sodium sulfate. Evaporation ofethyl acetate gave an oil which was chromatographed over silica gel(methylene chloride:methanol, 8:2). UV visible fractions were combinedand evaporated to give a thick oil which was dried under vacuum. Theproduct was crystallized from acetonitrile (172 mg), mp 163°-64° C. ¹ HNMR (DMSO d₆) δ 1.26 (s, 12H, CH₃), 1.89 (m, 2H, HNCH₂ CH₂ CH₂ NH), 2.12(m, 4H, HNCH₂ CH₂ CH₂ NH), 5.22 (s, 2H, CH₂ N<), 7.07 and 7.23 (s, 2H,imiH), 11.4 (s, 2H, OH). MS (FAB); (M+H)⁺ =495.

Analysis Calc'd for C₁₉ H₃₀ N₁₀ O₆.0.56 H₂ O: C, 45.22; H, 6.22; N,27.66; Found: C, 45.32; H, 6.09; N, 27.66.

Example 18 Synthesis of3,3,9,9-Tetramethyl-6-methoxy-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. 2-Methoxy-1,3-diaminopropane

N,N'-di-t-Boc-2-hydroxydiaminopropane was prepared as follows. To asolution of 1,3-diamino-2-hydroxy propane (25 g, 0.277 mole) in water(20 ml), di-tert-butyl dicarbonate (133 g, 0.61 mole) in THF (200 ml)was added followed by triethylamine (62 g, 0.7 mole) at 0° C. andstirred for 2 hours. The reaction mixture was allowed to come to roomtemperature and kept stirred for 16 hours more. Solvent THF andtriethylamine were removed under aspirator vacuum and the paste wasdiluted with water (250 ml). The solution was then thoroughly extractedwith ethyl acetate (5×100 ml) and the combined organic layer was washedwith water and brine. The dried ethyl acetate layer was concentratedunder reduced pressure to a gummy residue which was triturated withhexanes to yield a colorless solid. The solid was then recrystallizedfrom hexanes/ether.

Yield: 55.0 g (68%). m.p. 99°-101° C. ¹ H NMR (CDCl₃) δ 1.45 (s, 9H,t-C₄ H₉), 3.20 (m, 4H, HNCH₂ CHOHCH₂ NH), 3.72 (m, 1H, HNCH₂ CHOHCH₂NH), 5.05 (bs, 1H, NHCO).

Sodium hydride (2.4 g, 0.1 mol) was added in small portions to asolution of the product N,N'-di-t-Boc-2-hydroxypropanediamine (30 g.,103 mol) in dry THF (600 ml) over a period of 30 minutes. Methyl iodide(21.3 g, 0.15 mol) was added dropwise and the mixture stirred at roomtemperature for 6 hours. Additional methyl iodide (21.3 g, 0.15 mol) wasadded and the stirring was continued for further 6 hours. THF and excessmethyl iodide were removed on a rotary evaporator and the viscous oilobtained was chromatographed over silica gel (hexane:ethyl acetate 9:1).Fractions containing the N,N'-di-t-Boc-2-methoxy 1,3-diaminopropane werecollected and evaporated. The resultant oil solidified on standing. Itwas crystallized from hexane (17.2 g), mp 74°-75° C., ¹ H NMR (CDCl₃) δ1.45 (s, 9H, t-C₄ H₉), 3.05-3.35 (m, 4H, HNCH₂ CHOCH₃ CH₂ NH), 3.41 (s,3H, OCH₃), 5.05 (bs, 1H, NHCO).

N,N'-Di-t-boc-2-methoxy-1,3-diaminopropane (31.7 g, 0.1 mol) was addedto methanolic HCl (100 mL) and the solution was stirred at roomtemperature for 30 minutes. Methanol was removed on a rotary evaporatorand the residue was treated with methanolic ammonia to afford the titleA compound as a thick viscous oil (9.2 g). ¹ H NMR (D₂ O) δ 3.08-3.32(m, 4H, H₂ NCH₂ CHOCH₃ CH₂ NH₂), 3.31 (s, 3H, OCH₃), 3.52 (m, 1H, CH)

B. N-(3-Amino-2-methoxypropyl)-1-amino-1,1-dimethyl-2-butanone oxime

The title A compound (9.2 g, 0.091 mol) was dissolved in absolutemethanol (50 mL) and the solution was cooled to 0° C.3-Chloro-3-methyl-2-nitrosobutane (6.25 g, 0.04 mol, Example 1) wasadded over a period of 1 hour. The reaction mixture was stirred at 0° C.for further 1 hour and at room temperature for 12 hours. Methanol wasremoved on a rotary evaporator and the residue was dissolved indioxane-water (2:1, 300 mL) and the solution was cooled to 0° C. Sodiumcarbonate (21.2 g, 0.2 mol) was added to this solution, followed bydi-tert-butyl dicarbonate (42.0 g, 0.2 mol). The reaction mixture wasstirred at 0° C. for 1 hour and room temperature for 6 hours.Dioxane-water was removed on a rotary evaporator and the residue waspoured into water and extracted with ethyl acetate. The ethyl acetatelayer was washed with water and dried (Na₂ SO₄). This solution wasevaporated on a rotary evaporator and the residue was chromatographedover silica gel (hexane:ethyl acetate 50:50). N,N'-di-t-boc-2-methoxy-1,3-diaminopropane (formed from the unreacted2-methoxy-1,3-diaminopropane) eluted first. The t-boc derivative of theproduct was collected and evaporated to a thick oil which solidified onstanding. Yield 7.5 g (25%). ¹ H NMR (CDCl₃) δ 1.22 (s, 6H, CH₃), 1.42(s, 9H, t-C₄ H₉), 1.6 (bs, 1H, NH), 1.85 (s, 3H, CH₃ C═NOH), 2.52-3.28(m, 4H, HNCH₂ CHOCH₃ CH₂ NH₂), 3,41 (s, 3H, OCH₃), 4.12 (q, 1H, CH),5.35 (bs, 1H, NHCO).

The t-Boc derivative (7.5 g, 0.0035 mol) was dissolved in methanolic HCl(50 mL) and the solution was stirred at room temperature for 30 minutes.Anhydrous ether (300 mL) was added and the precipitated amine-oximehydrochloride was collected by filtration and dried under vacuum. Thesolid was dissolved in methanol and neutralized with methanolic ammonia.Methanol was removed on a rotary evaporator and the free base thusobtained was dried under vacuum (3.8 g). ¹ H NMR (D₂ O) δ 1.22 (s, 6H,CH₃), 1.81 (s, CH₃ C═NOH), 2.52-3.18 (m, 4H, HNCH₂ CHCHOCH₃ CH₂ NH₂),3.31 (s, 3H, OCH₃), 3.52 (m, 1H, CH).

C.3,3,9,9-Tetramethyl-6-methoxy-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime

Diisopropylethylamine (0.35 g, 0.0028 mol) was added to a slurry of thetitle B compound (0.5 g, 0.0025 mol) and3-chloro-3-methyl-1-(2-nitro-1H-imidazo-1-yl)-2-nitrosobutane (0.7 g,0.0028 mol, Example 1) in acetonitrile (5 mL), and the reaction mixturewas heated to 45° C., with stirring. A clear solution was formed in 15minutes. The reaction mixture was stirred at 45° C. for a further 1hour. Acetonitrile was removed on a rotary evaporator and the residuewas dried under vacuum. The viscous oil obtained was treated withmethanolic ammonia and methanol was removed under vacuum. The resultantoil was chromatographed over silica gel (methylene chloride:methanol8:2). UV visible fractions were collected and evaporated on a rotaryevaporator. The resultant solid was crystallized from acetonitrile (0.12g), mp 169°-70° C. MS: (M+H)⁺ calc'd: 414.2465; found: 414.2472.

Anal. calc'd for C₁₇ H₃₁ N₇ O₅ : C, 49.38; H, 7.56; N, 23.71; Found: C,49.70; H, 7.59; N, 23.73.

Example 19 Synthesis of ⁹⁹ Tc! Oxo4,4,10,10-tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-5,9-diazadodecane-3,11-dionedioximatol (3-)-N,N', N", N'"! technetium(V)

Ethylene glycol (150 μL) was added to a stirred solution of N(t-butyl)₄!TcOCl₄ ⁻ (59.9 mg, 0.120 mmoles) dissolved in 1.0 ml MeOH. This wasfollowed by the addition of 0.75M Na acetate in MeOH (1.5 mL) and4,4,10,10-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-5,9-diazadodecane-3,11-dionedioxime (70.6 mg, 0.178 mmoles, Example 3) which caused the solution toturn clear red-orange-brown. After 5 minutes the solvent was removed byrotary evaporation to give a viscous, red-orange, opaque oil. Theproduct was redissolved in methylene chloride, and this solution waswashed with water (2×2.5 mL), and then dried over Na₂ SO₄. This solutionwas evaporated by rotary evaporation to yield to a bright orange solid.The solid was redissolved in <1 ml CH₂ Cl₂, and the product purified bypassage through a silica gel column that was conditioned and eluted withdiethyl ether. The orange band was collected, and the solvent evaporatedto give a bright red solid and which was recrystallized from CH₂ Cl₂/hexane. The product was isolated by suction filtration, rinsed withhexane and dried in vacuo overnight. The yield of the product was 29.5mg as small, bright orange crystals. M.S.: (M+H)⁺ =510; (MH)⁻ =508.

Analysis calc;d. for C₁₇ H₂₈ N₇ O₅ Tc: C, 40.08; H, 5.54; N, 19.25;Found: C, 39.92; H, 5.84; N, 19.15.

Example 20 Synthesis of ^(99m) TC! Oxo3,3,6,6,9,9-hexamethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioximato!(3-)-N,N',N",N'"! technetium(V), by reaction in aqueousethanol

3,3,6,6,9,9-Hexamethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioxime (3.08 mg, Example 14) was dissolved in EtOH (1 mL). 0.1M Aqueoussodium bicarbonate solution (0.5 mL) and ^(99m) TcO₄ ⁻ in saline (0.8mL, 57.1 mCi) were added, followed by a saturated solution of stannoustartrate in saline (150 μL). The mixture was shaken, and allowed tostand at room temperature for 10 minutes.

Example 21 Synthesis of ^(99m) TC! Oxo3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazo-1-yl)-4,8-diazaundecane-2,10-dionedioximato! (3-)-N,N',N",N'"! technetium(V) using SnDTPA as the reducingagent

A kit containing 2 mg of the ligand in Example 1 in a lyophilized formwas prepared. A vial of the above lyophilized formulation wasreconstituted with saline and ^(99m) Tc-generator eluate, such that thetotal reconstitution volume was 2 mL and the radioactive concentrationadjusted as required. A vial of a commercially-available kit containing500 μg of stannous chloride and 10 mg of DTPA was reconstituted with 2mL of saline. 100 μL of the stannous DTPA solution was transferred tothe above reconstituted kit of the ligand in Example 1. The vial wasshaken and allowed to stand at room temperature for 10 minutes. Theradiochemical purity of the product was assayed by HPLC, and determinedto be >95%.

The results of the further compounds tested accordance with Example 8are summarized below.

    ______________________________________                                        Compound name/number                                                                           E.sub.pc (V)                                                                          Reduction process                                    ______________________________________                                        Compound from Ex. 2                                                                            -1.81   reversible                                           Compound from Ex. 3                                                                            -1.54   reversible                                           Compound from Ex. 4                                                                            -1.51   reversible                                           Compound from Ex. 5                                                                            -1.52   reversible                                           Compound from Ex. 6a                                                                           -1.81   reversible                                                            -2.02   irreversible                                         Compound from Ex. 6b                                                                           -1.48   reversible                                                            -1.96   irreversible                                         Compound from Ex. 19                                                                           -1.53   reversible                                                            -2.02   irreversible                                         ______________________________________                                    

Example 23 Synthesis of3,3,6,9,9-Pentamethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. Preparation of 2-Methyl malonamide

Dimethyl methylmalonate (100.0 g, 0.68 mol) in dry methanol (500 mL) wassaturated with dry ammonia gas at 0° C. and the reaction mixture wasstirred at room temperature for 24 hours. The colorless solid whichseparated was isolated by filtration, washed and dried to yield thetitle product; yield: 77.0 g (96%); mp. 209°-210° C.; ¹ H NMR (DMSO-d₆)δ 1.34 (d, 3H, CH₃), 3.18 (q, 1H, CH), 7.05 & 7.35 (2bs, 4H, CONH₂).

B. Preparation of 1,3-Diamino-2-methylpropane

Borane in tetrahydrofuran (THF) (1M, 1100 mL) was added from a syringeto a suspension of 2-methyl malonamide (25.0 g, 215.5 mmol) in THF (50mL) over a period of 30 minutes, then stirred at 40° C. under nitrogenatmosphere for 24 hours. The reaction mixture was then cooled to 0° C.HCl (2N, 40 mL) was added and the mixture was stirred for 30 minutes.The solvent was removed on a rotary evaporator and the semi-solid thusobtained was co-evaporated with dry methanol (5×20 mL) to remove boricacid. After neutralization with 1N NaOH, the resulting oil was distilledunder reduced pressure to provide the title product as a colorless oil.

Yield: 13.65 g (72%); bp. 90°-92° C./108-110 mm. ¹ H NMR (CDCl₃) δ 0.96(d, 3H, CH₃), 1.50 (m, 1H, CH), 2.72 (q, 2H, NCH₂), 2.55 (q, 2H, NCH₂).

C. Preparation of3-(3-Amino-2-methyl-propylamino)-3-methyl-1-(2-nitroimidazolyl)-2-butanoneoxime

3-Chloro-3-methyl-1-(2-nitroimidazolyl)-2-nitrosobutane (1.5 g, 6.09mmol, Example 1) was added portionwise over a period of 30 minutes to astirred solution of 1,3-diamino-2-methylpropane (3.0 g, 34.10 mmol) indry acetonitrile (30 mL) at 50° C. After the addition the reactionmixture was allowed to remain at 50° C. for an additional 30 minutes.The reaction mixture was then cooled and solvent was removed on a rotaryevaporator to a give a paste. This was crystallized fromdichloromethane-ether to afford the title product as a light yellowsolid; yield: 1.36 g (75%); mp. 102°-104° C. (decomp); ¹ H NMR (CDCl₃) δ0.89 (d, 3H, CH₃), 1.22 s, 6H, C(CH₃)₂ !, 1.51 (m, 1H, CH₃ CH), 2.25 (d,2H, CH₂ NH₂), 2.65 (m, 2H, NHCH₂), 5.31 (s, 2H, imi-CH₂), 7.03 & 7.21(2s, 2H, imi-H). MS m/e 299 (M+H)⁺.

D. Preparation of3,3,6,9,9-pentamethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane2,10-dione dioxime

3-Chloro-3-methyl-2-nitrosobutane (1.0 g, 7.38 mmol, Example 1) wasadded in portions to a stirred solution of 3-(3-amino-2-methylpropylamino)-3-methyl-1-(2-nitroimidazolyl)-2-butanone oxime (1.0 g, 3.36mmol) and diisopropylethylamine ((i-Pr)₂ NEt) (0.48 g, 3.72 mmol) in dryacetonitrile (20 mL) at 50° C. under nitrogen atmosphere. After theaddition, the reaction mixture was stirred for an additional 2 hours at50° C. and cooled to room temperature. The solvent was removed on arotary evaporator to yield a paste which was repeatedly crystallizedfrom acetonitrile to afford the title product as a cream colored solid;yield: 1.10 g (83%); mp. 167°-168° C. ¹ H NMR (DMSO-d₆) δ 0.8 (d, 3H,CH₃), 1.31 s, 6H, C(CH₃)₂ !, 1.58 2s, 6H, C(CH₃)₂ !, 1.89 s, 3H,C(N═OH)CH₃ !, 2.28 (m, 1H, CH₃ CH), 2.62 & 2.92 (2m, 4H, NHCH₂), 5.31(s, 2H, imi-CH₂), 7.06 & 7.22 (2S, 2H, imi-H) and 10.82 & 11.69 (2s, 2H,NOH). MS m/e 398 (M+H)⁺.

Anal. Calcd for C₁₇ H₃₁ N₇ O₄.0.89H₂ O: C, 47.37; H, 8.12; N, 22.75.Found: C, 47.37; H, 7.85; N, 22.41.

Example 24 Synthesis of12-Methoxycarbonyl-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,10-dionedioxime

A. Preparation of 5-Methyl-4-hexenonitrile

A solution of 5-bromo-2-methyl-2-pentene (9.5 g, 58.28 mmol) in drydimethylformamide (DMF) (10 mL) was treated with NaCN (5.0 g, 102.04mmol) and stirred at 80° C. for 15 hours. The reaction mixture was thenpoured into water and extracted with ethyl acetate (3×50 mL). Thecombined organic layer was washed with water (2×50 mL), dried andevaporated to provide the title product as a light brown oil (5.5 g,84%) which was used in the next step without further purification ¹ HNMR (CDCl₃) δ 1.62 & 1.75 2s, 6H, C(CH₃)₂ !, 2.32 m, 4H, (CH₂)₂ !, and5.11 (t, 1H, CH). MS m/e 127 (M+NH₄)⁺.

B. Preparation of 5-Methyl-4-hexenoic acid

5-Methyl-4-hexenonitrile (5.5 g, 50.46 mmol) was treated with NaOH (5N,50 mL) and heated at 110° C. for 15 hours. After acidification withconcentrated HCl to pH 2.0, the solution was extracted with ethylacetate (3×50 mL). The organic layer was then washed with water (2×50mL), dried and evaporated under vacuum to afford a brown liquid. Thiswas purified by vacuum distillation. The title product was obtained as acolorless liquid in 70% (4.52 g) yield; b.p. 75°-77° C./0.5 mm. ¹ H NMR(CDCl₃) δ 1.65 & 1.73 2s, 6H, C(CH₃)₂ !, 2.38 m, 4H, (CH₂)₂ !, 5.08 (t,1H, CH) and 10.45 (bs, 1H, COOH).

C. Preparation of Methyl 5-methyl-4-hexenoate

Freshly prepared diazomethane in ether was added to an ice-cooledsolution of 5-methyl-4-hexenoic acid (4.5 g, 35.16 mmol) in ether (10mL) until the solution became slightly yellow in color. The solvent wasthen evaporated under vacuum to afford the title product as a colorlessliquid in near quantitative yield (4.90 g). This product was used in thenext reaction step without further purification. ¹ H NMR (CDCl₃) δ 1.62& 1.68 2s, 6H, C(CH₃)₂ !, 2.30 m, 4H, (CH₂)₂ !, 3.69 (s, 3H, COOCH₃) and5.07 (t, 1H, CH).

D. Preparation of Methyl 5-chloro-4-nitroso-5-methylhexanoate

A mixture of isoamyl nitrite (12 mL) and methyl 5-methyl-4-hexenoate(4.90 g, 35.00 mmol) was cooled to -15° C. To this stirred solution,concentrated HCl (12.5 mL) was added dropwise from an addition flaskmaintaining the temperature of the reaction mixture below -5° C. Afterthe addition, the reaction mixture was stirred at 0° C. for 30 minutes,filtered and the precipitate washed with ethanol (-10° C.). The lightblue colored solid thus obtained was dispersed in petroleum ether (50mL), cooled to ˜-50° C. and filtered to provide the title product as acolorless solid. Yield: 3.0 g (41.3%); m.p. 90°-91° C. ¹ H NMR (CDCl₃) δ1.68 & 1.71 2s, 6H, C(CH₃)₂ !, 2.35 m, 4H, (CH₂)₂ !, 3.73 (s, 3H,COOCH₃) and 5.11 (t, 1H, CH). MS m/e 208 (M+H)⁺.

E. Preparation of3-(Aminopropyl)-3-methyl-1-(2-nitroimidazolyl!-2-butanone oxime

3-Chloro-3-methyl-1-(2-nitroimidazolyl)-2-nitrosobutane (1.00 g, 4.06mmol, Example 1) was added in portions over a period of 20 minutes to astirred solution of 1,3-diaminopropane (1.0 g, 13.50 mmol) in dryacetonitrile at 50° C. After the addition, the reaction mixture wasmaintained at 50° C. for an additional 30 minutes. The reaction mixturewas then cooled and filtered to provide the title compound as a yellowcolored solid. For further purification, the solid was suspended in asmall amount of water, stirred and filtered to afford the title productin >97% purity; yield: 0.78 g, (68%); mp. 136°-137° C. ¹ H NMR (DMSO-d₆)δ 1.19 s, 6H, C(CH₃)₃ !, 1.37 (m, CH₂ CH₂ CH₂), 1.52 (bs, 1N, NH), 2.21& 1.54 (2t, 4H, NHCH₂), 5.22 (s, 2H, imida-CH₂), 7.05 & 7.27 (2s, 2H,imida-ring H) and 11.42 (bs, 1H, NOH). MS m/e 285 (M+H)⁺.

F. Preparation of12-methoxycarbonyl-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,10-dionedioxime

Methyl 5-chloro-5-methyl-4-nitrosohexanoate (0.40 g, 1.92 mmol) wasadded to a suspension of3-(aminopropyl)-3-methyl-1-(2-nitroimidazolyl)-2-butanone oxime (0.50 g,1.76 mmol) and N,N-diisopropylethylamine (0.26 g, 2.02 mmol) in dryacetonitrile (25 mL), and the mixture was stirred at room temperaturefor 5 hours under nitrogen atmosphere. The solvent was removed on arotary evaporator and the paste thus obtained was triturated withdichloromethane to afford the title product as a light yellow coloredsolid (0.8 g), which was purified by crystallization from acetonitrile;mp. 137°-139° C.; ¹ H NMR (DMSO-d₆) δ 1.27 s, 6H, C(CH₃)₂ !, 1.48 s, 6H,C(CH₃)₂ !, 1.78 (m, 2H, NH), 2.51 & 2.79 2s & m, 10H, NH(CH₂)₃ NH,(CH₂)₂ COOCH₃ !, 3.62 (s, 3H, COOCH₃), 5.29 (s, 2H, imi-CH₂), 7.10 &7.29 (2s, 2H, imi-H) and 11.35 & 11.71 (2s, 2H, NOH). MS m/e 456 (M+H)⁺.Anal. Calcd for C₁₉ H₃₃ N₇ O₆.0.98H₂ O: C, 48.36; H, 7.42; N, 20.78.Found: C, 48.18; H, 7.31; N, 20.69.

Example 25 Synthesis of1-Ethoxy-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. Preparation of 1-Ethoxy-3-methyl-2-butene

Freshly prepared silver oxide (101 g, 0.37 mol) was added to a mixtureof 3-methyl-2-buten-1-ol (21.0 g, 25 mL, 0.24 mol) and ethyl iodide (300mL) and the stirred mixture was heated in an oil bath at 45° C. for 6hrs. Silver salts were removed by filtration and the filter cake waswashed with ether (250 mL). The filtrate and the washings were combinedand evaporated to remove ether and excess ethyl iodide. The oil thusobtained was distilled under atmospheric pressure to yield 14.8 g (51%)of tht title product as a colorless liquid. bp 119°-120° C. ¹ H NMR(CDCl₃) δ 1.2 (t, 3H, CH₂ CH₃), 1.72 (d, 6H, CH₃), 3.45 (m, 2H, CH₂CH₃), 3.95 (d, 2H, CH₂), 5.38 (t, 1H, (CH₃)₂ C═CH--).

B. Preparation of 3-Chloro-3-methyl-1-ethoxy-2-nitrosobutane

Concentrated HCl was added to a cooled (0°-5° C.) solution of isoamylnitrite (14.0 g, 0.12 mol) and 1-ethoxy-3-methyl-2-butene (6.84 g, 0.06mol). The temperature was maintained below 5° C. during the addition andthe reaction mixture was stirred at 5° C. for an additional 30 min. Theproduct was filtered and washed with a cold (-20° C.) 1:1 mixture ofethanol and ether. The solid was further washed with ether to afford thetitle product as a white solid. Yield 6.9 g (64%). mp: 84°-85° C. ¹ HNMR (CDCl₃) δ 1.12 (t, 3H, CH₂ CH₃), 1.65 (d, 6H, CH₃), 3.49 and 3.95(m, 2H, CH₂ OCH₂ CH₃), 4.15 (m, 2H, CH₂ CH₃), 6.12 (dd, 1H, CH₃)₂C═CH--!. MS: 180 (M+H)⁺.

C. Preparation of11-ethoxy-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)4,8-diazaundecane-2,10-dione dioxime

3-Chloro-3-methyl-1-ethoxy-2-nitrosobutane (180 mg, 1.0 mmol) was addedto a suspension of3-(aminopropyl)-3-methyl-1-(2-nitroimidazolyl)-2-butanone oxime (280 mg,1.0 mmol, Example 24) and N,N-diisopropylethylamine (130 mg, 1.0 mmol)in dry acetonitrile (15 mL), and the reaction mixture was stirred atroom temperature for 6 hours under nitrogen atmosphere. The solvent wasthen removed on a rotary evaporator and the thick oil obtained wastriturated with dichloromethane to afford the title product. This waspurified by column chromatography (silica gel, CH₂ Cl₂ : CH₃ OH), andcrystallization from acetonitrile. mp. 122°-23° C. ¹ H NMR (D₂ O) δ 1.12(t, 3H, CH₂ CH₃), 1.32 and 1.45 s, 12H, C(CH₃)₂ !, 1.82 (m, 2H, NHCH₂CH₂ CH₂ NH), 2.68 and 2.90 (m, 4H, NHCH₂ CH₂ CH₂ NH), 3.55 (s, 3H, OCH₂CH₃), 4.29 (s, 2H, CH₂ OCH₂ CH₃) 5.29 (s, 2H, CH₂), 7.10 and 7.24 (s,2H, imiH). HRMS: Calcd. (M+H)⁺ =428.2622⁺ ; Found: (M+H)⁺ =428.2624⁺.Anal. calcd. for: C₁₈ H₃₃ N₇ O₅ ; C, 50.57; H, 7.78, N, 22.93; Found: C,51.00, H, 7.80; N, 22.48.

Example 26 Synthesis of 3,3,9,9-Tetramethyl-6- 2-hydroxy-3-(2-nitro-¹H-imidazol-1-yl)propyl!-4,8-diazaundecane-2,10-dione dioxime

A. Preparation of 2-Allyl-malonamide

Diethyl 2-allyl-malonate (100 g, 0.649 mole) was dissolved in methanol(500 mL) and the solution was cooled 0° C. in ice bath. Gaseous ammoniawas bubbled into the solution to saturation while the solution wascooled in an ice bath. The reaction mixture was sealed and stirred atroom temperature for 20 hours. Solvent and ammonia were removed byrotary evaporation, and the residue was washed with ether to give thetitle product as a white solid. This was used for the next step withoutfurther purification. Yield: 76.5 g (92%). mp: 168°-169° C. 1H NMR(DMSO) δ 2.4 (m, 2H, CH₂ CH═CH₂); 3.0 (t, 1H, COCHCO); 5.0 (m, 2H, CH₂CH═CH₂); 5.7 (m, 1H, CH₂ CH═CH₂); 7.0 (bs, 2H, CONH₂); 7.3 (bs, 2H,CONH₂).

B. Preparation of 2-Allyl-N,N'-di-t-Boc-1,3-propanediamine

To a slurry of lithium aluminum hydride (11.8 g, 0.3 mol) indimethoxyethane (300 mL) was added a warm solution of 2-allyl-malonamide(14.2 g, 0.1 mole) in dimethoxyethane (800 mL) over a period of 1 hour.The reaction mixture was stirred at 50° C. for 48 hours. Excess lithiumaluminum hydride was destroyed by the addition of 10% NaOH and water andthe mixture was stirred at room temperature for 2 hours.Di-t-butyldicarbonate (50.0 g, 0.23 mole) was added and the reactionmixture was stirred for 24 hours. The reaction mixture was filtered, andthe filter cake washed with CH₂ Cl₂ (250 mL). The filtrate and thewashings were collected and evaporated on a rotary evaporator to afforda thick viscous oil. This oil was purified by column chromatography(silica gel, hexane:ethyl acetate, 9:1). Fractions containing theproduct were collected, combined, and evaporated to give a thick oilwhich solified on standing. Trituration with pentane gave the titleproduct as a white solid. mp: 84°-87.5° C. ¹ H NMR (CDCl₃) δ 1.4 (s,18H, t-Boc), 1.7 (m, 1H, CH), 2.85 and 3.22 (m, 4H, CH(CH₂ NH)), 5.0 (m,2H, CH₂ ═C), 5.75 (m, 1H, --CH═CH₂). MS: (M+H)⁺ =315.

C. Preparation of 2-(2,3-Epoxypropyl)-N,N'-di-t-Boc-1,3-propanediamine

m-Chloroperbenzoic acid (5.0 g, 0.022 mol) was added portionwise to acooled (0° C.) solution of 2-allyl-N,N'-di-t-Boc-1,3-propanediamine (5.0g, 0.016 mol) in CH₂ Cl₂ (30 mL), and the solution was stirred for 24hours. The precipitated m chlorobenzoic acid was removed by filtrationand the filtrate was taken up in ether (200 mL). Excessm-chloroperbenzoic acid was decomposed by the addition of sodium sulfitesolution (20%, 10 mL). The ether layer separated and was washed with asaturated solution of sodium bicarbonate, water and dried (Na₂ SO₄).Evaporation of ether gave the title product as a viscous oil which wasused in the next step without further purification. Yield: 5.2 g (98%).¹ H NMR (CDCl₃) δ 1.5 (m, 3H, CH and CH₂ CH--), 2.45 and 2.80 (m, 2H,epoxide), 3.0-3.4 (m, 5H, 1H epoxide and (CH₂ NHtBoc)₂), 5.0 and 5.5 (m,2H, NHtBoc). MS: (M+H)⁺ =330.

D. Preparation of2-(2-Hydroxy-3-(2-nitroimidazolyl)-propyl)-N,N'-di-t-Boc-1,3-propanediamin

2-(2,3-Epoxypropyl)-N,N'-di-t-Boc-1,3-propanediamine (1.0 g, 0.003 mol)was added to a mixture of 2-nitroimidazole (500 mg) and potassiumcarbonate (70 mg) in ethanol (50 mL). The mixture was refluxed undernitrogen for 12 hours. The reaction mixture was cooled and filtered. Theprecipitate was washed with water and the off-white solid thus obtainedwas air dried. The crude product was chromatographed over silica gel(CH₂ Cl₂ :CH₃ OH 95:5). The UV visible fractions were collected andevaporated to give the title product as a white solid, which was used inthe next step without further purification. Yield: 420 mg (28%). ¹ H NMR(DMSO) δ 1.2 (m, 2H, CHCH₂ CHOH--), 1.75 (m, 1H, CHCH₂ CHOH--), 2.9 (m,4H, CH(CH₂ NHtBoc)₂), 3.9 (m, 1H, CHOH), 4.1-4.4 (m, 2H, CHOHCH₂ N), 5.0(m, 1H, OH), 6.65 (m, 2H, NH), 7.1- and 7.52 (s, 2H, imi.H). MS: (M+H)⁺=443.

E. Preparation of2-(2-Hydroxy-3-(2-nitroimidazolyl)-propyl)-1,3-propanediamine

2-(2-Hydroxy-3-(2-nitroimidazolyl)-propyl)-N,N'-di-t-Boc-1,3-propanediamine(0.9 g, 0.002 mole) was treated with methanolic HCl (5 mL) and themixture was stirred for 30 minutes at room temperature. Methanol wasremoved on a rotary evaporator and the residue was neutralized withmethanolic ammonia. The methanolic solution was concentrated on a rotaryevaporator and the residue was dried under vacuum to yield the titleproduct as a light yellow solid. This solid was used in the next stepwithout further purification. Yield: 0.38 g (80.0%). ¹ H NMR (D₂ O) δ1.6 (m, 2H, CH₂ CHOHCH₂ N<), 2.2 (m, 2H, CH(CH₂ NH), 2.98 (m, 2H, CH₂CHOHCH₂ N<) 4.05 (m, 1H, CHOH), 4.3-4.55 (m, 2H, CHOHCH₂ N<), 7.1 and7.32 (s, 2H, NCH═CH).

F. Preparation of3,3,9,9-Tetramethyl-6-(2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)-n-propyl)-4,8-diazaundecane-2,10-dione

2-Bromo-2-methylbutan-3-one (400 mg, 0.0024 mol, Example 5) was added tomixture of the2-(2-hydroxy-3-(2-nitroimidazolyl)-propyl)-1,3-propanediamine (240 mg,0.001 mole) and sodium bicarbonate (200 mg, 0.00245 mol) in DMF (2 mL),and the mixture was stirred at 45° C. for 6 hours. DMF was removed underreduced pressure and the residue was triturated with methylene chloride(3×5 mL). Methylene chloride was removed on a rotary evaporator and theoil thus obtained was chromatographed over silica gel (CH₂ Cl₂ :methanol, 8:2). UV visible portions were collected and evaporated togive the title product diketone as an oil. Yield 100 mg (25%). ¹ H NMR(CDCl₃) δ 1.52 d, 12H, C(CH₃)₂ !, 2.12 (s, 6H, CH₃ >═O), 2.35-3.12 (m,5H, CH(CH₂ NH), 4.1 (m, 1H, CHOH ), 4.3-4.6 (m, 2H, CHOHCH₂ N<), 7.1 and7.42 (s, 2H, NCH═CH).

G. Preparation of 3,3,9,9-Tetramethyl-6-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propyl!-4,8-diazaundecane-2,10-dionedioxime

O-Trimethylsilylhydroxylamine (1 mL) was added to a solution of3,3,9,9-tetramethyl-6-(2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)-n-propyl)-4,8-diazaundecane-2,10-dione(10) mg, 0.00025 mole) in methylene chloride (1.0 mL) and the reactionmixture was stirred at room temperature for 24 hours. Methanol was addedto the reaction mixture and the resultant oil was isolated andchromatographed over silica gel (CH₂ Cl₂ : methanol, 9:1). UV visibleportions were collected and evaporated to give the product. This wasdissolved in minimum amount of water and freeze dried. The freeze driedsolid was recrystallized from acetonitrile to yield the title product.Yield 45 mg (25%). mp 185°-87° C. ¹ H NMR (D₂ O) δ 1.22 d, 12H, C(CH₃)₂!, 1.80 (s, 6H, CH₃ >═N), 2.8 (m, 5H, CH(CH₂ NH), 3.82 (m, 1H, CROH),4.2-4.58 (m, 2H, CHOHCH₂ N<), 7.1 and 7.32 (s, 2H, NCH═CH). MS: Calcdfor C₁₉ H₃₆ NO₅ 442.2778 (M+H)⁺. Found. 442.2781.

Example 27 Synthesis of3,3,9,9-Tetramethyl-1-(4-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. Preparation of1-(4-Methyl-2-nitro-1H-imidazol-1-yl)-3-methyl-2-butene and1-(5-Methyl-2-nitro-1H-imidazol-1-yl)-3-methyl-2-butene

3,3-Dimethylallyl bromide (14.08 g, 94.49 mmol) was added to asuspension of anhydrous K₂ CO₃ (13.0 g, 94.20 mmol) and4-methyl-2-nitroimidazole (10.0 g, 78.74 mmol, D. P. Davies et al, J.Heterocyclic Chem., 1982, 19, 253-256) in dry acetone (100 mL). Thereaction mixture was stirred at room temperature under nitrogenatmosphere for 15 hours. The insoluble inorganic material was removed byfiltration and the filtrate was evaporated to afford a brown paste whichwas loaded onto a silica gel column and eluted with hexane ethyl acetate(8.5:1.5). The fractions containing the compound with R_(f) 0.51 (silicagel TLC, hexane-ethyl acetate, 7:3) were collected and evaporated toafford a solid which was recrystallized from ethyl acetate-hexane toprovide 1-(4-methyl-2-nitro-1H-imidazol-1-yl)-3-methyl-2-butene as alight yellow crystalline solid in 60% (9.21 g) yield; mp. 53°--54° C; ¹H NMR (CDCl₃) δ 1.80 s, 6H, C(CH₃)₂ !, 2.26 (s, 3H, imi-CH₃), 4.97 (d,2H, J=7.26 Hz, NCH₂), 5.36 (t, 1H, J=7.26 Hz, CH₂ CH) and 6.90 (s, 1H,imi-CH). ¹³ C NMR (CDCl₃) δ 13.80 (imi-CH₃), 18.16 & 25.70 C(CH₃)₂ !,47.76 (NCH₂), 117.35 (CH₂ CH), 122.77 (imi-CH), 138.09 C(CH₃)₂ !, 139.97(imi-CCH₃) and 143.53 (CNO₂). MS m/e 196 (M+H)⁺.

The fractions containing the compound with R_(f) 0.41 (silica gel,hexane-ethyl acetate 7:3) were pooled and evaporated to provide a solidwhich was recrystallized from ethyl acetate-hexane to afford1-(5-methyl-2-nitro-1H-imidazol-1-yl)-3-methyl-2-butene as a lightyellow fluffy solid. Yield: 1.02 g (15.2%); mp. 78°-79° C.; ¹ H NMR(CDCl₃) δ 1.75 & 1.82 2s, 6H, C(CH₃)₂ !, 2.32 (s, 3H, imi-CH₃), 4.97 (d,2H, J=6.60 Hz, NCH₂), 5.13 (t, 1H, J=6.55 Hz, CH₂ CH) and 6.97 (s, 1H,imi-CH). ¹³ C NMR (CDCl₃) δ 10.35 (imi-CH₃), 18.28 & 25.62 C(CH₃)₂ !,45.20 (NCH₂), 117.69 (CH₂ CH), 127.07 (imi-CH), 134.49 C(CH₃)₂ !, 137.57(imi-CCH₃) and 145.52 (CNO₂). MS m/e 196 (M+H)⁺.

B. Preparation of3-Chloro-3-methyl-2-nitroso-1-(4-methyl-2-nitro-1H-imidazol-1-yl)butane

Concentrated HCl (20 mL) was added from an addition flask to a mixtureof 1-(4-methyl-2-nitro-1H-imidazol-1-yl)-3-methyl-2-butene (5.0 g,25.64) and isoamyl nitrite (20 mL), keeping the temperature in the rangeof 0° C. to 5° C. After the addition the reaction mixture was stirred atice-cold temperature for 30 minutes. The solid which formed was removedby filtration and washed with ice-cold ethanol. The solid was dispersedin acetonitrile, cooled 0° C. and filtered to provide the title productas a light blue colored solid; yield: 4.97 g (62%); mp. 110°-113° C.(decomp); ¹ H NMR (DMSO-d₆) δ 1.81 s, 6H, C(CH₃)₂ !, 2.16 (s, 3H,imi-CH₃), 5.36 (s, 2H, NCH₂), 7.12 (s, 1H, imi-CH) and 11.98 (s, 1H,NOH). MS m/e 261 (M+H)⁺.

C. Preparation3,3,9,9-Tetramethyl-1-(4-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioxime

3-Chloro-3-methyl-2-nitroso-1-(4-methyl-2-nitro-1H-imidazol-1-yl)butane(3.41 g, 13.09 mmol) was added in portions to a stirred solution ofN-(3-aminopropyl)-1-amino-1,1-dimethyl-2-butanone oxime (2.0 g, 11.56mmol, Example 1) and (i-Pr)₂ NEt (1.64 g, 13.09 mmol) in dryacetonitrile (20 mL) at 50° C. under a nitrogen atmosphere. After theaddition the reaction mixture was stirred for an additional 2 hours at50° C. and cooled to room temperature. The solid which formed wasfiltered and recrystallized from acetonitrile to provide the titleproduct as a light yellow colored solid.

Yield: 3.08 g (59.4%); mp. 140°-141° C.; ¹ H NMR (DMSO-d₆) δ 1.12 & 1.102s, 6H, C(CH₃)₂ !, 1.33 (m, 2H, CH₂ CH₂ CH₂), 1.70 s, 3H, C(═N)CH₃ !,1.79 (bs, 2H, NH), 2.15 (s, 3H, imi-CH₃), 2,19 (m, 4H, NHCH₂), 5.19 s,2H, NCH₂ C(═N)!, 7.05 (s, 1H, imi-CH) and 10.33 & 11.36 (2s, 2H, NOH).MS m/e 398 (M+H)⁺.

Example 28 Synthesis of3,3,9,9-Tetramethyl-1-(5-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioxime

A. Preparation of3-Chloro-3-methyl-2-nitroso-1-(5-methyl-2-nitro-1H-imidazol-1-yl)butane

3-Chloro-3-methyl-2-nitroso-1-(5-methyl-2-nitro-1H-imidazol-1-yl)butanewas prepared in 60% yield from1-(5-methyl-2-nitro-1H-imidazol-1-yl)-3-methyl-2-butene (1.0 g, 5.12mmol, Example 27), isoamyl nitrite (5.0 mL) and concentrated HCl (5.0mL) following the procedure described for the preparation of itspositional isomer (Example 27B) as a light blue solid; mp. 102°-105° C.(decomp); ¹ H NMR (DMSO-d₆) δ 1.73 s, 6H, C(CH₃)₂ !, 2.32 (s, 3H,imi-CH₃), 5.53 (s, 2H, NCH₂), 6.97 (s, 1H, imi-CH) and 11.58 (s, 1H,NOH). MS m/e 261 (M+H)⁺.

B. Preparation of3,3,9,9-Tetramethyl-1-(5-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioxime

3,3,9,9-Tetramethyl-1-(5-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioxime was synthesized in 58% (0.66 g) yield as a yellow crystallinesolid from3-chloro-3-methyl-2-nitroso-1-(5-methyl-2-nitroimidazolyl)butane (0.75g, 2.88 mmol), N-(3-aminopropyl)-1-amino-1,1-dimethyl-2-butanone oxime(0.45 g, 2.60 mmol, Example 1) and (i-Pr)₂ NEt (0.37 g, 28.62 mmol) indry acetonitrile (10 mL) at 50 C. following the method described for thepreparation of its positional isomer (Example 27C); mp. 150°-152° C.; ¹H NMR (DMSO-d₆) δ 1.01 & 1.13 2s, 6H, C(CH₃)₂ !, 1.40 (m, 2H, CH₂ CH₂CH₂), 1.68 (bs, 2H, NH), 1.71 s, 3H, C(═N)CH₃ !, 2.25 (s, 3H, imi-CH₃),2.20 (m, 4H, NHCH₂), 5.36 s, 2H, NCH₂ C(═N)!, 6.91 (s, 1H, imi-CH) and10.36 & 11.06 (2s, 2H, NOH). MS m/e 398 (M+H)⁺.

Example 29 Synthesis of the ^(99m) Tc Complexes of the Ligands ofExamples 23 to 28

The ^(99m) Tc complexes of the ligands prepared in Examples 23 to 28above were prepared as follows:

The ligand prepared as the title compound of the Example (9 μmoles; 3.6to 4.1 mg, the latter depending on the molecular weight of the ligand)was dissolved in methanol (0.1 ml) in a 5 ml glass vial, and 0.1M NaHCO₃buffer (0.5 ml), 0.9% saline, and ⁹⁹ Mo/^(99m) Tc generator eluate(total saline/eluate volume=1.4 ml) were added. The vial was sealed, anda saturated solution of stannous tartrate in saline (50 μl) was added tothe vial. The vial was shaken to mix the reagents, and allowed to standat room temperature for 3 minutes. The radiochemical purities (RCP) ofthe ^(99m) Tc complexes were measured by reversed phase HPLC asdescribed above in Example 7a. All ligands labeled to give RCPvalues >94% within 3 minutes after mixing. The complexes so obtainedfrom the ligands of Examples 23 to 28 were, respectively:

Oxo3,3,6,9,9-pentamethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioximato!(3-)-N,N',N",N'"!technetium-^(99m) Tc(V);

Oxo12-methoxycarbonyl-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,10-dionedioximato!(3-)-N,N',N",N'"!technetium-^(99m) Tc(V);

Oxo(11-ethoxy-3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioximato!(3-)-N,N',N",N'"!technetium-⁹⁹ Tc(V);

Oxo 3,3,9,9-tetramethyl-6-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propyl!-4,8-diazaundecane-2,10-dionedioximato!(3-)-N,N',N",N'"!technetium-^(99m) Tc(V);

Oxo3,3,9,9-tetramethylol-(4-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioximato!-(3-)-N,N',N",N'"!technetium-^(99m) Tc(V); and

Oxo3,3,9,9-tetramethyl-1-(5-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioximato!-(3-)-N,N',N",N'"!technetium-^(99m) Tc(V).

Example 306,6-Difluoro-3,3,9,9-tetramethyl-12-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,10-dionedioxime

A. 1,2-Diamino-2,2-difluoropropane dihydrochloride

To a suspension of 2,2-difluoromalonamide (10 g, 90%, 65.22 mmol) in drytetrahydrofuran (THF) (20 mL) was added BH₃ -THF (350 mmol, 1M, 350 mL)dropwise from a syringe over a period of 30 minutes. The reactionmixture was then stirred at 45° C. under nitrogen atmosphere for 24hours. To the ice-cold reaction mixture HCl (2N, 50 mL) was added andthe reaction mixture was stirred for 30 minutes. The solvent was thenremoved on a rotary evaporator and the semi-solid thus obtained wasdissolved in water (100 mL), and treated with di-t-butyl dicarbonate(32.0 g, 147.8 mmol) in dioxane (200 mL) and Na₂ CO₃ (30.0 g, 280 mmol).After stirring overnight at ambient temperature, the reaction mixturewas concentrated (50 mL), and extracted with ethyl acetate (4×75 mL).The combined extracts gave a colorless solid on evaporation undervacuum. For further purification, the compound (impregnated with silicagel) was loaded onto a silica gel column and eluted with a hexane-ethylacetate (7:3) solvent mixture. The fractions with compound werecollected and evaporated to provide 1,3-bis-N-t-butyloxycarbonyl2,2-difluoropropanediamine as a colorless crystalline solid in 48% yield(9.70 g); TLC silica gel, hexane-ethyl acetate (6:4)!R_(f) 0.54; mp.125°-125° C.; ¹ H NMR (CDCl₃) d 1.45 s, 18H, C(CH₃)₃ !, 3.52 (m, 4H,NHCH₂) and 5.26 (bt, 2H, NH). MS m/e 311 (M+H)⁺.

A solution of 1,3-bis-N-t-butyloxycarbonyl 2,2-difluoropropanediamine(4.5 g, 14.52 mmol) in CH₃ OH (5 mL) was treated with methanolic HCl (5mL) at 0° C. and stirred for 20 minutes. The removal of the volatilesunder vacuum afforded a colorless crystalline solid of1,3-diamino-2,2-difluoropropane as a hydrochloride salt in nearquantitative yield (4.85 g); mp. 187°-190° C.; ¹ H NMR (D₂ O) d 3.62 (t,J_(HF) =16.20 Hz, 4H, NHCH₂). MS m/e 111 (M+H)⁺.

B.4-(3-Amino-2,2-difluoropropylamino)-4-methyl-1-(2-nitro-1H-imidazol-1-yl)-3-pentanoneoxime

To a mixture of 1,3-diamino-2,2-difluoropropane dihydrochloride (title Adiamine) (1.10 g, 6.01 mmol) and N,N-diisopropylethylamine (2.72 g,21.04 mmol) in acetonitrile (10 mL) at 40° C. was added4-chloro-4-methyl-1-(2-nitro-1H-imidazol-1-yl)-3-nitrosopentane (Example3, step (B)) (0.50 g, 1.90 mmol) in small portions with 30 stirringunder nitrogen atmosphere. After the addition, the stirring wascontinued for an additional 30 minutes and the solvent was removed on arotary evaporator. The paste obtained was treated with silica gel andthe silica gel powder impregnated with the compound was loaded onto asilica gel column and eluted with CH₃ OH--CH₂ Cl₂ (5:95). The fractionscontaining compound were collected and evaporated to give the titleproduct as a light yellow colored solid. Yield: 0.42 g (66%), mp.112°-114° C.; ¹ H NMR (CDCl₃) d 1.19 s, 6H, C(CH₃)₂ !, 2.88 m, 4H,C(═N)CH₂ & NHCH₂ !, 3.71 (t, 3H, J_(HF) =13.19 Hz, CH₂ NH₂), 4.72 (t,2H, J=7.26, CH₂ CH₂ N), 7.11 & 7.26 (2s, 2H, nitroimid-H) and 10.79 (s,1H, NOH). MS m/e 335 (M+H)⁺.

C.6,6-Difluoro-3,3,9,9-tetramethyl-12-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,0-dionedioxime

A clear solution of4-(3-amino-2,2-difluoropropylamino-4-methyl-1-(2-nitro-1H-imidazol-1-yl)-3-pentanoneoxime title B compound) (0.40 g, 1.20 mmol) andN,N-diisopropylethylamine (0.42 g, 3.32 mmol) in acetonitrile (5 mL) wastreated with 3-chloro-3-methyl-2-nitrosobutane (prepared analogously to3-chloro-3-methyl-2-nitroisobutane, Example 1, step (B)) (0.45 g, 3.32mmol) with stirring at room temperature under nitrogen atmosphere for 2hours. After evaporation of the solvent under vacuum, the paste thusobtained was loaded, as a powder adsorbed on silica gel, onto a silicagel column and eluted with methanol-dichloromethane (2:98). Thefractions with compound were pooled together and evaporated to affordthe title product, which was further purified by crystallization fromCH₂ Cl₂ --CH₃ OH to a cream colored solid. Yield: 0.45 g (89%); mp.90°-92° C.; ¹ H NMR (DMSO-d₆) d 1.12 s, 12H, C(CH₃)₂ !, 1.70 s, 3H,C(═NOH)CH₃ !, 2.18 & 2.22 (2t, 2H, NH), 2.62 (m, 4H, CH₂ CF₂), 2.80 (t,2H, J=7.25 Hz, CH₂ CH₂ N), 4.61 (t, 2H, J=7.25 Hz, CH₂ CH₂ N), 7.15 &7.52 (2s, 2H, imi-H) and 10.46 & 10.90 (2s, 2H, NOH). MS m/e 434 (M+H)⁺.Anal. Calcd for C₁₇ H₂₉ N₇ O₄ F₂ : C, 47.11; H, 7.74; N, 22.62; F, 8.77.Found: C, 47.28; H, 6.54; N, 22.79; F, 8.53. HPLC: Retention time: 20.74min Microsorb-C₁₈, 0.46×25 cm, 5 m; solvent: 0.1% trifluoroacetic acid(TFA)/water (A) and 0.1% TFA/acetonitrile (B); flow rate: 1.0 mL/min.;run condition: linear gradient, 1% increase in B per min.; ran at 230and 254 nm for 50 min.; in both cases a single peak was observed; 98.95%(230 nm) and 100% (254 nm)!.

Example 31 3,3,9,9-Tetramethyl-1-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propoxy!-4,8-diazaundecane-2,10-dionedioxime

A. 3,3-Dimethylallylglycidyl ether

To a solution of dimethylallyl alcohol (17.3 g, 20.5 mL, 0.2 mol) in dryTHF (200 mL), sodium hydride (4.8 g, 0.2 mol) was added in portions andthe mixture was stirred at room temperature for 1 hour. Epibromohydrin(27.4 g, 17.12 mL, 0.2 mol) was added to this reaction mixture dropwiseand the mixture was stirred at room temperature for 24 hours. THF wasremoved on a rotary evaporator and the residue was taken up in ether andfiltered. The ether solution was concentrated on a rotary evaporator andthe brown oil obtained was distilled under vacuum to yield the titleproduct. bp. 93°-94° C./10 mm. Yield: 17.2 g (60.5%). ¹ H NMR (CDCl₃) δ1.68 and 1.75 (s, 6H, CH₃), 2.61 and 2.88 (dd, 2H, oxirane CH₂), 3.17(m, 1H, oxirane CH), 3.38 and 3.7 (m, 2H, CH₂ OCH₂ CH), 4.05 (m, 2H, CH₂OCH₂ CH), 5.35 (m, 1H, >C═CH).

B. 1- 2-Hydroxy-3-(2-nitro-1H-imidazol-1-yl)-propyldimethylallyl ether

To a mixture of 3,3-dimethylallylglycidyl ether (title A epoxide) (9.0g, 0.063 mol) and 2-nitroimidazole (7.2 g, 0.063 mol) in ethanol (75mL), potassium carbonate (0.75 g, 0.005 mol) was added and the mixturewas refluxed in an oil bath for 4 hours. The reaction mixture was cooledand poured into water. The yellow solid which formed was filtered andrecrystallized from aqueous ethanol to yield the title product. Yield:12.2 g, (76%), mp: 72°-73° C. ¹ H NMR (CDCl₃) δ 1.62 and 1.78 (s, 6H,CH₃), 2.78 (d, 1H, OH), 3.4 and 3.58 (m, 2H, CHOHCH₂ O), 4.0 (d, 1H,CHOH), 4.40 and 4.68 (m, 2H, CHOHCH₂ N) 5.35 (m, 1H, >C═CH), 7.1 and 7.3(s, 2H, imiH). Anal. calcd. for C₁₈ H₁₇ N₃ O₄ : C, 51.76; H, 7.71; N,16.46. Found: C, 51.60; H, 6.48; N, 16.42.

C. 3-Chloro-1-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl!propoxy!-3-methyl-2-nitrosobutane

To a cooled (0°-5° C.) stirred slurry of 1-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propyl-dimethylallyl ether (7.0 g,0.0275 mol) in isoamyl nitrite (43 g, 50 mL, 0.042 mol) was addedconcentrated hydrochloric acid (2.5 mL, 0.03 mol) with stirring. Thereaction mixture was maintained below 5° C. during the addition andstirred at 5° C. for an additional 2 hrs. The solid formed was stirredwith cold ether-ethanol (3:1, 150 mL), filtered and dried under vacuumto yield the title product. Yield: 5.8 g (67%). mp: 116°-117° C. dec. ¹H NMR (DMSO) δ 1.55 and 1.62 s, 6H, CH₃ !, 3.35 (m, 4H, CH₂ OCH₂ CHOH),3.82 (m, 1H, CHOH), 4.1-4.52 (m, 2H, CHOHCH₂ N<), 5.3 (m, 1H, CHOH), 6.0(dd, 1H, CHNO), 7.15 and 7.42 (s, 2H, imi H).

D. 3,3,9,9-Tetramethyl-1-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propoxy!-4,8-diazaundecane-2,10-dionedioxime

A slurry of 3-chloro-1-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propoxy!-3-methyl-2-nitrosobutane(3.2 g, 0.01 mol) in acetonitrile (35 mL) was maintained at 40° C. for30 minutes. To this slurry was added a solution ofN-(3-aminopropyl)-1-amino-1,1-dimethyl-2-butanoneoxime (1.73 g, 0.01mol) in acetonitrile (10 mL) and diisopropylethylamine (1.5 g, 0.012mol) and the mixture was maintained at 40° C. for 48 hrs. The solidwhich formed was filtered and the acetonitrile solution was evaporatedon a rotary evaporator to give a green viscous oil which was dried undervacuum. The foamy solid obtained was triturated with acetonitrile toafford a thick oil which solidified on standing. This was purified bycolumn chromatography (CH₂ Cl₂ :CH₃ OH, 7:3, 50:50)). The fractionscontaining the product were collected and evaporated to give an oilwhich was dried under vacuum and triturated with acetonitrile to give awhite solid. It was recrystallized from ethyl acetate to yield the titleproduct. Yield: 1.2 g (26%). mp: 134°-35° C. MS: (M+H)+=458⁺. ¹ H NMR(DMSO) δ 1.05 and 1.16 s, 12H, C(CH₃)₂ !, 1.38 (m, 2H, NHCH₂ CH₂ CH₂NH), 1.72 (s, 3H, CH₃), 2.2 (m, 2H, NHCH₂ CH₂ CH₂ NH), 3.38 (m, 4H, CH₂OCH₂ CHOH), 3.8 (m, 2H, OCH₂ CHOH), 4.28 and 4.6 (m, 3H, CHOHCH₂ N<),7.12 and 7.58 (s, 2H, imi H), 10.4 and 10.85 (s, 2H, NOH). Anal. Calcdfor C₁₉ H₃₅ N₇ O₆ : C, 49.88; H, 7.71; N, 21.43. Found: C, 49.80; H,7.79; N, 21.47.

Example 326-Hydroxy-3,3,9,9-tetramethyl-12-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,10-dionedioxime

A. 4-Methyl-1-(2-nitro-1H-imidazol-1-yl)-3-pentene

5-Bromo-2-methyl-2-pentene (25 g, 0.154 mol) was dissolved in drydimethylformamide (DMF) (200 mL). To the solution was added K₂ CO₃ (21.3g, 0.154 mol) and 2-nitroimidazole (17.4 g, 0.154 mol). The mixture wasstirred under N₂ atmosphere at 75° C. for 48 hours. DMF was evaporatedon a rotary evaporator. The yellow gummy residue was stirred with water(150 mL) to give yellow solid, which was dissolved in diethyl ether (150mL), dried over Na₂ SO₄ and evaporated on a rotary evaporator to yieldthe title product. Yield 27.8 g (92%). mp: 49°-51° C. MS: (M+H)⁺ =196,M⁺ =195. ¹ H NMR (CDCl₃) δ 1.45 and 1.68 (s, 6H, gem-di-CH₃), 2.52 (q,2H, CH₂ CH═), 4.43 t, 2H, CH₂ -(2-nitroimidazolyl)!, 5.08 (t, 1H, CH₂CH═), 7.05 and 7.14 (s, 2H, 2-nitroimidazolyl-H).

B. 4-Chloro-4-methyl-1-(2-nitro-1H-imidazol-1-yl)-3-nitrosopentane

4-Methyl-1-(2-nitro-1H-imidazol-1-yl)-3-pentene (8 g, 41 mmol) wasdissolved in isoamyl nitrite (50 mL) at room temperature. The solutionwas cooled to 0° C. in an ice-salt bath and concentrated HCl (12 mL) wasadded dropwise. The reaction temperature was maintained between 3°-5° C.during the HCl addition; the reaction was stirred in an ice-salt bathfor 45 minutes after the addition of HCl. The product was filtered,washed with ethanol-ether (1:2) and dried in vacuum to give 8.6 g (81%)of the title product as a white solid; mp: 96°-97° C. MS: (M+H)⁺ =261,M⁺ =260. ¹ H NMR (DMSO-d₆) δ 1.70 (s, 6H, gem-di-CH₃), 2.94 (t, 2H, CH₂C═NOH), 4.65 t, 2H, CH₂ -(2-nitroimidazole)!, 7.16 and 7.52 (s, 2H,2-nitroimidazolyl-H), 11.43 (s, 1H, CH₂ C═NOH).

C. 3- 3-Amino-2-hydroxy!propylamino-3-methyl-2-oximinobutane

To a stirring solution of 1,3-diamino-2-hydroxypropane (9.0 g, 0.1 mol)in acetonitrile (100 mL), was added anhydrous potassium carbonate (14.0g 0.1 mol). The mixture was cooled to and3-chloro-3-methyl-2-nitrosobutane (13.5 g, 0.1 mol) (prepared accordingto E. G. Vassian et al., Inorg. Chem., 1967, 2043-2046) was added inportions over a period of 2 hours. After the addition, the reactionmixture was stirred at room temperature for 2 hours and then heatedunder reflux for 6 hours. The mixture was cooled and filtered and washedwith acetonitrile. The combined organic layer was concentrated to apaste and treated with saturated methanolic HCl (100 mL). The solutionwas again concentrated to a paste and then crystallized from methanoltwice to yield the title product as a colorless HCl salt. Yield: 10.5 g(48%). m.p.>185° C. (dec.). ¹ H NMR (D₂ O) δ 1.45 (s, 6H, C--CH₃), 1.8(s, 3H, N═C--CH₃), 3.0 (m, 4H, N--CH₂) and 4.1 (m, 1H, O--CH).

D.6-hydroxy-3,3,9,9-tetramethyl-12-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,10-dionedioxime

3- 3-Amino-2-hydroxy!propylamino-3-methyl-2-oximinobutane hydrochloride(5.4 g, 0.25 mol) was neutralized with methanolic ammonia and evaporatedto paste which was dried under vacuum for 2 hours at room temperature.The dried free base was suspended with stirring in dry acetonitrile (50mL), treated with anhydrous potassium carbonate (3.5 g, 0.025 mol) andwarmed to about 60° C. the above warm solution,4-chloro-4-methyl-1-(2-nitro-1H-imidazol-1-yl)-3-nitrosopentane (7.0 g,0.26 mol) was added all at once with stirring; the reaction temperaturewas maintained at 60° C. After the monooxime starting material haddisappeared (16-20 hours), the mixture was filtered and washed withacetonitrile. The combined acetonitrile solution was concentrated to apaste and then chromatographed on a flash column. Elution with 85:15EtOAc--CH₃ OH yielded the product as a yellow foam. The title productwas repeatedly crystallized at -20° C. from a mixture of ethyl acetate(EtOAc)-acetonitrile until a single peak was observed on HPLC analysis.Yield: 4.0 g (91% pure, 25%). Yield of the pure product (99%): 0.3 g.m.p. 62°-64° C. ¹ H NMR (DMSO-d₆) δ 1.5 (s, 12 H, C--CH₃), 2.1 (s, 3H,N═C--CH₃), 2.7 (m, 4H, N--CH₂), 3.2 (t,2H, N═C--CH₂), 3.9 (bs, 1H,O--CH), 5.0 t, 2H, imid-CH₂), 7.45 (s, 1H, imid-H), 8.0 (1H, imid-H),10.9 (s, 1H, N--OH) and 11.2 (s, 1H, N--OH). HRMS: Calcd for C₁₇ H₃₂ N₇O₅, 414.2476; Found, 414.2465. Anal. Calcd for C₁₇ H₃₁ N₇ O₅.0.1 EtOAcC, 47.98; H, 7.87; N, 23.04. Found C, 48.48; H, 7.59; N, 22.54.

Example 334,4,10,10-Tetramethyl-1,13-bis(2-nitro-1H-imidazol-1-yl)-5,9-diazatridecane-3,11-dionedioxime

To a solution of 1,3-diaminopropane (1.46 g, 15 mmol) in dryacetonitrile (50 mL) was added anhydrous potassium carbonate (4.2 g, 30mmol) and the solution was brought to 50°-60° C.4-Chloro-4-methyl-1-(2-nitroimidazol-1H-yl-3-nitrosopentane (3.9 g, 30mmol, Example 3B) was added as a solid and the reaction mixture wasstirred for 20 hours. The cooled solution was filtered and thoroughlywashed with dry acetonitrile. The insoluble solid was ground to a powderand suspended in water with stirring. The water insoluble portion wasfiltered, washed with water and air dried, avoiding direct exposure tolight. The resulting bright yellow solid was recrystallized fromacetonitrile to yield the title product as a pale yellow solid. Yield:2.6 g (33%). m.p. 101°-103° C. (dec.). ¹ H NMR (DMSO-d₆) δ 1.48 (s, 12H,C-methyls), 1.78 (m, 2H, N--CH₂ --CH₂ --CH₂ --N), 2.58 (t, 4H,N═C--CH₂), 3.21 (t, 4H, N--CH₂), 4.98 (t, 4H, imi-CH₂), 7.58 (s, 2H,imi-H), 7.96 (s, 2H, imi-H) and 11.20 (s, N--OH). M. S. M+H!⁺ 524. HRMSFound: 523.2748; Calcd. 523.2741. Anal. Calc. C₂₁ H₃₄ N₁₀ O₆ 0.56 H₂ O:C, 47.35; H, 6.65; N, 26.29. Found: C, 47.67; H, 6.48; N, 25.97.

Example 34 Synthesis of the ^(99m) Tc Complexes of the Ligands ofExamples 30 to 33

The ^(99m) Tc complexes of the ligands prepared in Examples 30 to 33above were prepared as follows:

Ligand (2-4 mg) was dissolved in 0.9% NaCl saline (1.0 mL) and 0.1M HCl(0.1 mL) in a 5 mL glass vial, and 0.1M sodium hydrogen carbonate buffer(0.5 mL), saline, and ⁹⁹ Mo/^(99m) Tc generator eluate (totalsaline/eluate volume=0.5 mL) was added. The vial was sealed, and asaturated solution of stannous tartrate in saline (50 μL) was added tothe vial. The vial was shaken to mix the reagents, and allowed to standat room temperature. The radiochemical purities (RCP) of the ^(99m) Tccomplexes were measured by reversed phase HPLC as described above. Alltechnetium complexes formed with RCP>94% after 3 minutes, except for the^(99m) Tc complex of the ligand of Example 32, which formed more slowly(at 5 minutes the RCP was 68%; at 91 minutes the RCP had increased to86%). The complexes so obtained from the ligands of Examples 30 to 33were, respectively:

Oxo6,6-Difluoro-3,3,9,9-tetramethyl-12-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,10-dionedioximato!(3-)-N,N',N",N'"!technetium-^(99m) Tc(V);

Oxo 3,3,9,9-Tetramethyl-1-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propoxy!-4,8-diazaundecane-2,10-dionedioximato!(3-)-N,N',N",N'"!technetium-^(99m) Tc(V);

Oxo6-hydroxy-3,3,9,9-tetramethyl-12-(2-nitro-1H-imidazol-1-yl)-4,8-diazadodecane-2,10-dionedioximato!(3-)-N,N',N",N'"!-technetium-^(99m) Tc(V); and

Oxo4,4,10,10-tetramethyl-1,13-bis(2-nitro-1H-imidazol-1-yl)-5,9-diazatridecane-3,11-dionedioximato!(3-)-N,N',N",N'"!technetium-^(99m) Tc(V).

What is claimed is:
 1. A method for the diagnostic imaging of hypoxictissue in a mammalian species comprising the steps of:administering apharmaceutical formulation comprising a metal complex, said metalcomplex comprising a radionuclide of technetium and a ligand of eitherformula Ia or Ib: ##STR46## wherein at least one R' is --(A)_(p) --R₂where (A)_(p) is a linking group and R₂ is a nitro-heterocyclic hypoxialocalizing moiety; and wherein the other R' groups, that are not--(A)_(p) --R², and the R groups are the same, or different and areindependently selected from hydrogen, halogen, hydroxy, alkyl, alkonyl,alkynyl, alkoxy, aryl, --COOR₃, ##STR47## --NH₂, hydroxyalkyl,alkoxyalkyl, hydroxyaryl, haloalkyl, arylalkyl, alkyl--COOR₃,--alkyl--CON(R₃)₂, --alkyl--N(R₃)₂, --aryl--COOR₃, --aryl--CON(R₃)₂,--aryl--N(R₃)₂, 5- or 6-membered nitrogen- or oxygen-containingheterocycle; or two groups taken together with the one or more atoms towhich they are attached form a carbocyclic or heterocyclic, saturated orunsaturated spiro or fused ring which may be substituted with R groups;R₁ is hydrogen, a thiol protecting group or --(A)_(p) --R₂ ; R₃ ishydrogen, alkyl or aryl; m=2 t5; p=0 t20; and where the term aryldenotes phenyl or substituted phenyl, andobtaining a diagnostic image ofsaid hypoxic tissue.
 2. The method of claim 1 used to diagnose ischemictissue in the heart.
 3. The method of claim 1 used to diagnose ischemictissue in the lung.
 4. The method of claim 1 used to diagnose ischemictissue in the kidneys or liver.
 5. The method of claim 1 used todiagnose ischemic tissue in the brain.
 6. The method of claim 1 used todiagnose hypoxic tissue in tumors.
 7. The method of claim 1 wherein theligand has the name 3,3,9,9-tetramethyl-1-2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propoxy)-4,8-diazaundecane-2,10-dionedioxime.
 8. The method of claim 1 wherein the ligand has the name3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioxime.